High quantum yield acridinium compounds and their uses in improving assay sensitivity

ABSTRACT

The present invention relates to hydrophilic, high quantum yield acridinium compounds. It has been discovered that the placement of electron-donating groups in the acridinium ring system increases the amount of light that is emitted by the corresponding acridinium compound when its chemiluminescence is triggered by alkaline peroxide. More specifically, it has been found that the placement of one or two hydrophilic, alkoxy groups at the C-2 and/or C-7 position of the acridinium ring system of acridinium compounds increases their quantum yield and enhances the aqueous solubility of these compounds. The present hydrophilic, high quantum yield, acridinium compounds are useful chemiluminescent labels for improving the sensitivity of immunoassays.

This application is a divisional of application Ser. No. 11/142,938filed Jun. 2, 2005 which is a continuation-in-part of application Ser.No. 10/260,504 filed Sep. 27, 2002, both of which are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to high quantum yield chemiluminescentacridinium compounds with increased light output. Structural featuresnecessary for obtaining increased light emission from acridiniumcompounds are disclosed herein. Additionally, we also disclosehydrophilic versions of these structures, which not only have increasedlight output but also have increased water solubility and lownonspecific binding. These compounds because of their enhanced quantumyield and hydrophilic nature, are useful in improving assay sensitivity.

2. Background of the Invention

Chemiluminescent acridinium esters (AEs) are extremely useful labelsthat have been used extensively in immunoassays and nucleic acid assays.A recent review, Pringle, M. J., Journal of Clinical Ligand Assay vol.22, pp. 105-122(1999) summarizes past and current developments in thisclass of chemiluminescent compounds.

McCapra, F. et al., Tetrahedron Lett. vol. 5, pp.3167-3172 (1964) andRahut et al. J. Org. Chem vol. 301, pp. 3587-3592. (1965) disclosed thatchemiluminescence from the esters of acridinium salts could be triggeredby alkaline peroxide. Since these seminal studies, interest inacridinium compounds has increased because of their utility as labels.The application of the acridinium ester9-carboxyphenyl-N-methylacridinium bromide in an immunoassay wasdisclosed by Simpson, J. S. A. et al., Nature vol. 279, pp. 646-647(1979). However, this acridinium ester is quite unstable, therebylimiting its commercial utility. This instability arises from hydrolysisof the 9-carboxyphenyl ester linkage between the phenol and theacridinium ring.

Different strategies for increasing the stability of acridiniumcompounds have been described in the prior art. Law et al., Journal ofBioluminescence and Chemiluminescence, vol. 4, pp. 88-89 (1989)introduced two methyl groups to flank the acridinium ester moiety tostabilize this linkage. The resulting sterically stabilized acridiniumester, DMAE-NHS [2′,6′-dimethyl-4′-(N-succinimidyloxycarbonyl)phenyl10-methylacridinium-9-carboxylate] was found to have the same lightoutput as an acridinium ester lacking the two methyl groups. Thestability of the former compound when conjugated to an immunoglobulinwas vastly superior and showed no loss of chemiluminescent activity evenafter one week at 37° C. at pH 7. In contrast, the unsubstitutedacridinium ester only retained 10% of its activity when subjected to thesame treatment. U.S. Pat. Nos. 4,918,192 and 5,110,932 describe DMAE andits applications.

The sterically-stabilized acridinium ester, DMAE-NHS has been usedcommercially in the ACS:180™ immunoanalyzer (Bayer Diagnostics). U.S.Pat. No. 5,656,426 to Law et al. discloses a hydrophilic version of DMAEtermed NSP-DMAE-NHS ester. Both DMAE and NSP-DMAE are currently used inBayer's ACS:180™ and Advia Centaur™ immunoanalyzers. The chemicalstructures of these compounds and the numbering system of the acridiniumring are illustrated in the following figures:

Because the acridinium ring is symmetrical, C-1 is equivalent to C-8,C-2 is equivalent to C-7, C-3 is equivalent to C-6, and C-4 isequivalent to C-5.

U.S. Pat. No. 6,664,043 B2 to Natrajan et al discloses NSP-DMAEderivatives with hydrophilic modifiers attached to the phenol. Thestructure of one such compound is illustrated in the above figure. Inthis compound a diamino hexa(ethylene) glycol (diamino-HEG) moiety isattached to the phenol to increase the aqueous solubility of theacridinium ester. A glutarate moiety was appended to the end of HEG andwas converted to the NHS ester to enable labeling of various molecules.

A different class of stable chemiluminescent acridinium compounds hasbeen described by Kinkel et al., Journal of Bioluminescence andChemiluminescence vol. 4, pp. 136-139 (1989) and Mattingly, Journal ofBioluminescence and Chemiluminescence vol. 6, pp. 107-114 (1991) andU.S. Pat. No. 5,468,646. In this class of compounds, the phenolic esterlinkage is replaced by a sulfonamide moiety, which is reported to imparthydrolytic stability without compromising the light output. Inacridinium esters, the phenol is the ‘leaving group’ whereas inacridinium sulfonamides, the sulfonamide is the ‘leaving group’ duringthe chemiluminescent reaction with alkaline peroxide.

Light emission from acridinium compounds is normally triggered byalkaline peroxide. The overall light output, which can also be referredto as the chemiluminescence quantum yield, is a combination of theefficiencies of the chemical reaction leading to the formation of theexcited-state acridone and the latter's fluorescence quantum yield.

A number of factors can influence the overall light output of acridiniumcompounds. The intrinsic chemiluminescence quantum yields of acridiniumcompounds are markedly affected by their structures. While most studieshave focused on the effect of the leaving group on light emission, nonehave addressed the effect of functional groups on the acridinium ring onchemiluminescence quantum yields although their effects on thewavelengths of light emission have been well documented. See U.S. Pat.No. 6,355,803. Although, the synthesis of an acridinium ester withmethoxy groups at C-2 and C-7 of the acridinium ring system has alsobeen disclosed in U.S. Pat. No. 5,521,103, the effect of the two methoxygroups on either the quantum yield or wavelength of light emission ofthe acridinium ester was not disclosed.

DESCRIPTION OF THE DRAWINGS

In the accompanying drawings:

FIG. 1( a) is a graphical representation of the theophylline assay athigh theophylline concentrations;

FIG. 1( b) is a graphical representation of the theophylline assay atlow theophylline concentrations;

FIG. 2( a) is a graphical representation of the thyroid stimulatinghormone (TSH) assay at low TSH concentrations;

FIG. 2( b) is a graphical representation of the TSH assay at high TSHconcentrations.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to acridinium compounds with enhancedquantum yields. It has been discovered that the placement ofelectron-donating groups in the acridinium ring increases the amount oflight that is emitted by the corresponding acridinium compound when itschemiluminescence is triggered by alkaline peroxide. The concept ofelectron-donating groups is well known to practitioners in the field.Typically, an electron-donating group is a functional group, that is, anatom or a collection of atoms that, when compared to hydrogen, willdonate electrons. A detailed discussion on electron-donating andelectron-withdrawing groups can be found in Smith et al., AdvancedOrganic Chemistry: Reactions, Mechanisms and Structure, pp. 16-17(5^(th) Edition Wiley-Interscience).

More specifically, it has been found that the placement of one or twoelectron-donating functional groups, such as methoxy or alkoxy groups atC-2 and/or C-7 of the acridinium ring of acridinium compounds increasestheir quantum yield. Table 1 summarizes the relative quantum yields ofvarious methoxy and alkoxy substituted acridinium compounds in relationto NSP-DMAE as well as their emission wavelength maxima. The chemicalstructures of representative acridinium compounds with electron-donatingfunctional groups at the C-2 and/or C-7 position of the acridinium ringand corresponding acridinium compounds without electron-donatingfunctional groups at the C-2 and/or C-7 position of the acridinium ringare listed in Table 1 and are structurally represented thereafter.

The acridinium compounds listed in Table 1 were synthesized usingorganic chemistry techniques well known to practitioners in the field.The acridinium ester NSP-DMAE, which does not have any electron-donatinggroups in the acridinium nucleus, was used as a reference compound.Information about the synthesis of the compounds in Table 1 can be foundin the Examples. Light emission from each compound was measured using aluminometer equipped with a photo-multiplier tube as the detector andthe emission wavelength was measured using a FSSS (Fast SpectralScanning System) camera from Photoresearch Inc.

TABLE 1 Relative Quantum Yields of Acridinium Compounds EmissionRelative wavelength Compound quantum yield maximum, nm  1. NSP-DMAE 1426  2. NSP-2-OMe-DMAE 2.2 458  3 NSP-3-OMe-DMAE 0.35 418  4NSP-4-OMe-DMAE 0.33 478  5 NSP-2,4-(OMe)₂-DMAE 0.35 514  6.NSP-2,7-(OMe)₂-DMAE 2.7 484  7. NSP-2,7-(OSP)₂-DMAE 3.1 480  8.NSP-2,5-(OMe)₂-DMAE 0.5 486  9. NSP-2,4,7-(OMe)₃-DMAE 1 518 10.2,7-(OMHEG)₂-DMAE 1.5 480 11. NSP-2,7-(OMHEG)₂ DMAE 3.3 480 12. NSP-AS 1426 13. NSP-2,7-(OMe)₂-AS 1.7 484 Chemiluminescence was measured for 5seconds on a Magic Lite Analyzer Luminometer (MLA1, Bayer Diagnostics).For quantum yield measurements, samples of the various compounds wereprepared in 10 mM phosphate pH 8 containing 150 mM NaCl, 0.05% bovineserum albumin (BSA) and 0.01% sodium azide. For emission spectraldeterminations, samples were prepared in dimethyl formamide (DMF).

The chemical structures of the compounds listed in Table 1 are asfollows:

From an inspection of Table 1, it is evident that either a singlemethoxy group at C-2 or two methoxy groups at C-2 and C-7 of theacridinium ring, increase the light output from the correspondingacridinium compound. For example, NSP-2-OMe-DMAE has 2.2 times the lightoutput of NSP-DMAE while NSP-2,7-(OMe)₂-DMAE has 2.7 times (270%) thelight output of NSP-DMAE. Methoxy groups at other positions on theacridinium ring system do not lead to enhanced light emission. Thus, thefinding that the specific placement of methoxy groups on the acridiniumring is required to obtain enhanced light yield is unexpected.

In addition to methoxy groups, other alkoxy groups (“OR”), wherein R canbe alkyl, alkenyl, alkynyl, aryl, and aralkyl containing up to 20heteroatoms, at C-2 and C-7 of the acridinium ring also lead to enhancedlight emission. This finding is important because the structuralcomponents leading to enhanced light emission can be combined withhydrophilic groups to produce a hydrophilic, high quantum yieldacridinium compound. For example, replacement of the two methoxy groupsin NSP-2,7-(OMe)₂-DMAE with O-sulfopropyl groups (“OSP”) in the compoundNSP-2,7-(OSP)₂-DMAE also leads to enhanced light emission, that is, 3.1times the light output of NSP-DMAE. Besides the O-sulfopropyl group,other hydrophilic groups such as the O-methoxy hexa(ethylene) glycolgroups (“OMHEG”) also lead to enhanced light emission.

Thus the two compounds 2,7-(OMHEG)₂-DMAE and NSP-(OMHEG)₂-DMAE emit 1.5times and 3.3 times more light, respectively, than NSP-DMAE as shown inTable 1. In 2,7-(OMHEG)₂-DMAE, the acridinium nitrogen contains a methylgroup whereas NSP-(OMHEG)₂-DMAE contains a sulfopropyl moiety. Theseobservations clearly demonstrate that —OR groups in general can be usedas electron-donating groups at these positions to obtain enhanced lightemission. The key functional group substitution is thus the directplacement of electron-donating atoms at the C-2 and/or C-7 position onthe acridinium nucleus.

A further advantage of the O-sulfopropyl groups as well as the OMHEGgroups is that the resulting acridinium esters are extremely watersoluble owing to the three sulfonate moieties or the methoxyhexa(ethylene)glycol moieties in these compounds. Other poly(ethylene)glycol moieties besides hexa(ethylene)glycol (HEG) can also be used. Ahigh quantum yield acridinium ester that contains —OMTEG[O-methoxytri(ethylene)glycol] groups at C-2 and C-7 of the acridiniumring has also been synthesized as described in Example 10.

The acridinium sulfonamide with two methoxy groups at C-2 and C-7 alsoshows enhanced light emission in comparison to the unsubstitutedacridinium sulfonamide. For example, NSP-2,7-(OMe)₂-AS emits 1.7 timesmore light than NSP-AS under identical conditions. Since the acridiniumsulfonamide compounds contain a different leaving group than acridiniumesters, the enhanced light emission obtained from NSP-2,7-(OMe)₂-ASclearly indicates that the leaving group does not play a critical role.The enhancement of quantum yield due to the presence ofelectron-donating groups at the C-2 and/or C-7 positions of theacridinium ring applies to both acridinium esters and acridiniumsulfonamides.

The emission wavelength maxima of the various compounds listed in Table1 show that the placement of methoxy or alkoxy groups at every positionin the acridinium ring, except at C-3, leads to a shift to longerwavelength when compared to unsubstituted NSP-DMAE. However, thereappears to be no simple correlation between emission wavelength maximumand quantum yield. For example, NSP-4-OMe-DMAE has an emission maximumof 478 nm and yet its quantum yield is only one third that of NSP-DMAE.A further point to note is that the nature of the alkyl group attachedto the oxygen atoms at C-2 and C-7 does not appear to affect theemission wavelength maximum. Thus, NSP-2,7-(OMe)₂-DMAE;NSP-2,7-(OSP)₂-DMAE and NSP-2,7-(OMHEG)₂-DMAE all have the same emissionwavelength maxima.

In summary, Table 1 discloses acridinium compounds with specificstructural components, such as the presence of an —OR group wherein Rcan be alkyl, alkenyl, alkynyl, aryl, and aralkyl containing up to 20heteroatoms, at C-2 and/or C-7 of the acridinium ring, that contributeto enhanced light emission. A high quantum yield acridinium compound hasa relative light yield greater than that of NSP-DMAE which is used asthe reference compound in this study and is assigned a value of 1.

Acridinium compounds are used extensively in immunoassays and nucleicacid assays. Analytes that are typically measured in such assays areoften substances of some clinical relevance and can span a wide range ofmolecules from large macromolecules such as proteins, nucleic acids,viruses bacteria, and the like, to small molecules such as ethanol,vitamins, steroids, hormones, therapeutic drugs, and the like.

A ‘sandwich’ immunoassay typically involves the detection of a largemolecule, also referred to as macromolecular analyte, using two bindingmolecules such as antibodies. One antibody is immobilized or attached toa solid phase such as a particle, bead, membrane, microtiter plate orany other solid surface.

The methodology for the attachment of binding molecules such asantibodies to solid phases is well known in the prior art. For example,an antibody can be covalently attached to a particle containing amineson its surface by using a cross-linking molecule such as glutaraldehyde.The attachment may also be non-covalent and may involve simpleadsorption of the binding molecule to the surface of the solid phase,such as polystyrene beads and microtiter plate. The second antibody isoften covalently attached with a chemiluminescent or fluorescentmolecule often referred to as a label. Labeling of binding moleculessuch as antibodies and other binding proteins are also well known in theprior art and are commonly called conjugation reactions and the labeledantibody is often called a conjugate. Typically, an amine-reactivemoiety on the label reacts with an amine on the antibody to form anamide linkage. Other linkages such as thioether, ester, carbamate, andthe like between the antibody and the label are also well known in theprior art.

In the sandwich assay, the two antibodies bind to different regions ofthe macromolecular analyte. The macromolecular analyte can be proteins,nucleic acids, oligosaccharides, antibodies, antibody fragments, cells,viruses, receptors, or synthetic polymers. The binding molecules can beantibodies, antibody fragments, nucleic acids, peptides, bindingproteins or synthetic binding polymers. For example, the folate bindingprotein (“FBP”) binds the analyte folate. Synthetic binding moleculesthat can bind a variety of analytes have also been disclosed by Mossbachet al. Biotechnology vol. 14, pp. 163-170 (1996).

When the solid phase with the immobilized antibody and the labeledantibody is mixed with a sample containing the analyte, a bindingcomplex is formed between the analyte and the two antibodies. This typeof assay is often called a heterogeneous assay because of theinvolvement of a solid phase. The chemiluminescent or fluorescent signalassociated with the binding complex can then be measured and thepresence or absence of the analyte can be inferred. Usually, the bindingcomplex is separated from the rest of the binding reaction componentssuch as excess, labeled antibody prior to signal generation. For exampleif the binding complex is associated with a magnetic bead, a magnet canbe used to separate the binding complex associated with the bead frombulk solution.

By using a series of ‘standards’, that is, known concentrations of theanalyte, a ‘dose-response’ curve can be generated using the twoantibodies. Thus, the dose-response curve correlates a certain amount ofmeasured signal with a specific concentration of analyte. In a sandwichassay, as the concentration of the analyte increases, the amount ofsignal also increases. The concentration of the analyte in an unknownsample can then be calculated by comparing the signal generated by anunknown sample containing the macromolecular analyte, with thedose-response curve.

In a similar vein, the two binding components can also be nucleic acidsthat bind or hybridize to different regions of a nucleic acid analyte.The concentration of the nucleic acid analyte can then be deduced in asimilar manner.

In the assays described above, as the concentration of the analytedecreases, the amount of signal also decreases. At extremely low analyteconcentrations, the ability to distinguish the specific signal of thelabel molecule associated with the binding complex, from ‘noise’ arisingfrom non-specific binding of the labeled antibody to the solid phase,becomes increasingly difficult.

Non-specific binding is a common phenomenon and in assays it is measuredas the signal in the absence of any analyte and often arises when thelabeled-antibody binds to the solid phase in a random manner. To be ableto measure the specific signal arising from a small concentration ofanalyte, this specific signal must be greater in magnitude than thesignal associated with non-specific binding. Thus, to increase thesensitivity of an assay for an analyte, by which is meant the ability todetect and quantify very low amounts of an analyte, the specific signalmust be increased and non-specific binding must be reduced.

A common strategy for improving the sensitivity of assays that employchemiluminescent or fluorescent labels is to label one of the bindingmolecules with multiple labels in an attempt to increase the strength ofthe specific signal. However, this strategy has its own drawbacks suchas an increase in non-specific binding which negates the gain inspecific signal and moreover, multiple labeling of antibodies andnucleic acids can often have deleterious effects on their propertiessuch as their ability to bind analytes as well as their solubility. Forexample, multiple labeling of antibodies with hydrophobicchemiluminescent or fluorescent labels can cause aggregation orprecipitation of the antibody.

A more attractive approach to improving assay sensitivity is to enhancethe chemiluminescent or fluorescent property of the label of interest.Accordingly, one objective of the present invention is the improvementof assay sensitivity by the use of high quantum yield, acridiniumcompounds. Another objective of this invention is the disclosure ofhydrophilic, high quantum yield, acridinium compounds that not only haveincreased light output but are also extremely water-soluble andconsequently, have low non-specific binding properties. Suchchemiluminescent labels are unlikely to cause aggregation orprecipitation of proteins and nucleic acids when compared to hydrophobiclabels and offer a distinct advantage over conventional labels.

Finally, although multiple labeling strategies for signal amplificationusing hydrophobic labels are of limited utility, the present labelsbecause of their hydrophilic nature may be more suited to such anapproach. For example, a binding molecule such an antibody can be linkedto another molecule which can serve as the label ‘carrier’. In such acase, the binding molecule can be considered to be ‘indirectly labeled”through the carrier.

The second molecule or carrier can be a protein or synthetic moleculessuch as polymers such as polyamino acids, or dendrimers that can belabeled with the chemiluminescent compound. By employing such astrategy, the properties of the binding molecule are segregated andpreserved for the binding reactions, while the carrier molecule becomesthe signal bearer for large increases in signal.

Conjugation of proteins to other proteins and polymers are well known inthe prior art. A simple example of such an approach would be to label aprotein such as bovine serum albumin (BSA) with multiplechemiluminescent labels and subsequently, covalently attach or conjugatethe labeled BSA to an antibody for an analyte. The resulting labeledBSA-antibody conjugate when used in the assay is likely to generategreater amounts of signal by virtue of the multiple chemiluminescentlabels on the BSA.

Another class of molecules that can be particularly suitable as carriersare dendrimers that are commercially available from Dendritech Inc.These dendrimers, by virtue of their small size in relation to thenumber of functional groups they carry for the attachment of labels, canbe ideal carriers of chemiluminescent and fluorescent labels.

The attachment of the labeled carriers to the binding molecule orantibody can also be accomplished non-covalently. For example if theantibody molecule is labeled with biotin, then streptavidin with achemiluminescent label can bind to the biotinylated antibody because ofthe strong affinity of streptavidin for biotin. The resultingantibody-streptavidin conjugate can then be used in an assay for ananalyte.

Another class of immunoassays for small molecule analytes such assteroids, vitamins, hormones, therapeutic drugs or small peptidesemploys an assay format that is commonly referred to as a competitiveassay. Typically, in a competitive assay, a conjugate is made of theanalyte of interest and a chemiluminescent or fluorescent label bycovalently linking the two molecules. The small molecule analyte can beused as such or its structure can be altered prior to conjugation to thelabel.

The analyte with the altered structure is called an analog. It is oftennecessary to use a structural analog of the analyte to permit thechemistry for linking the label with the analyte. A structural analog ofan analyte is sometimes used to attenuate or enhance its binding to abinding molecule such an antibody. Such techniques are well known in theprior art. The antibody or a binding protein to the analyte of interestis often immobilized on a solid phase either directly or through asecondary binding interaction such as the biotin-avidin system describedearlier.

The concentration of the analyte in a sample can be determined in acompetitive assay by allowing the analyte-containing sample and theanalyte-label conjugate to compete for a limited amount of solidphase-immobilized binding molecule. As the concentration of analyte in asample increases, the amount of analyte-label conjugate captured by thebinding molecule on the solid phase decreases. By employing a series of‘standards’, that is, known concentrations of the analyte, adose-response curve can be constructed where the signal from theanalyte-label conjugate captured by the binding molecule on the solidphase is inversely correlated with the concentration of analyte. Once adose-response curve has been devised in this manner, the concentrationof the same analyte in an unknown sample can be determined by comparingthe signal obtained from the unknown sample with the signal in thedose-response curve.

Another format of the competitive assay for small molecule analytesinvolves the use of a solid phase that is immobilized with the analyteof interest or an analyte analog and an antibody or a binding proteinspecific for the analyte that is conjugated with a chemiluminescent orfluorescent label. In this format, the antibody-label conjugate iscaptured onto the solid phase through the binding interaction with theanalyte or the analyte analog on the solid phase.

The analyte of interest present in a sample then “competitively” bindsto the antibody-label conjugate and thus inhibits or replaces theinteraction of the antibody-label conjugate with the solid phase. Inthis fashion, the amount of signal generated from the antibody-labelconjugate captured on the solid phase is correlated to the amount of theanalyte in sample.

The high quantum yield acridinium compounds of the present inventionwhen used in competitive assays for small molecule analytes offer theadvantage of producing higher signals at all concentrations of analytethereby facilitating the measurement of a wider range of analyteconcentration. Thus, at high analyte concentration, when the amount oflabel-analyte conjugate captured by the binding molecule on the solidphase becomes very small, it is still possible to measure a discerniblesignal from a conjugate of a high quantum yield acridinium compound andthe analyte.

The hydrophilic, high quantum yield acridinium compounds of the presentinvention are useful for the detection of macromolecular analytes inheterogeneous assays with improved sensitivity comprising the followingsteps:

-   a) providing a conjugate of a binding molecule specific for a    macromolecular analyte with a hydrophilic, high quantum yield    chemiluminescent acridinium compound containing electron donating    functional groups at the C-2 and/or C-7 position of the acridinium    ring;-   b) providing a solid phase immobilized with a second binding    molecule specific for said macromolecular analyte;-   c) mixing the conjugate, the solid phase and a sample suspected of    containing the analyte to form a binding complex;-   d) separating the binding complex captured on the solid phase;-   e) triggering the chemiluminescence of the separated binding complex    by adding chemiluminescence triggering reagents;-   f) measuring the amount of light emission with a luminometer; and-   g) detecting the presence or calculating the concentration of the    analyte by comparing the amount of light emitted from the reaction    mixture with a standard dose response curve which relates the amount    of light emitted to a known concentration of the analyte.

The hydrophilic, high quantum yield acridinium compounds of the presentinvention are also useful for the detection of small molecule analytesin heterogeneous assays with improved sensitivity comprising thefollowing steps:

-   (a) providing a conjugate of an analyte with a hydrophilic, high    quantum yield chemiluminescent acridinium compound containing    electron donating functional groups at the C-2 and/or C-7 position    of the acridinium ring;-   (b) providing a solid phase immobilized with a binding molecule    specific for the analyte;-   (c) mixing the conjugate, solid phase and a sample suspected of    containing the analyte to form a binding complex;-   (d) separating the binding complex captured on the solid phase;-   (e) triggering the chemiluminescence of the separated binding    complex by adding chemiluminescence triggering reagents;-   (f) measuring the amount of light with an luminometer; and-   (g) detecting the presence or calculating the concentration of the    analyte by comparing the amount of light emitted from the reaction    mixture with a standard dose response curve which relates the amount    of light emitted to a known concentration of the analyte.

The hydrophilic, high quantum yield acridinium compounds of the presentinvention are also useful for the detection of small molecule analytesin heterogeneous assays with improved sensitivity comprising thefollowing steps:

-   a) providing a solid phase immobilized with an analyte or an analyte    analog;-   b) providing a conjugate of a binding molecule specific for the    analyte with a hydrophilic, high quantum yield chemiluminescent    acridinium compound containing electron donating functional groups    at the C-2 and/or C-7 position of the acridinium ring;-   c) mixing the solid phase, the conjugate and a sample suspected    containing the analyte to form a binding complex;-   (d) separating the binding complex captured on the solid phase;-   (e) triggering the chemiluminescence of the separated binding    complex by adding chemiluminescence triggering reagents;-   (f) measuring the amount of light with an luminometer; and-   (g) detecting the presence or calculating the concentration of the    analyte by comparing the amount of light emitted from the reaction    mixture with a standard dose response curve which relates the amount    of light emitted to a known concentration of the analyte.

The chemiluminescence triggering reagents can be either hydrogenperoxide or peroxide salts.

The utility of hydrophilic, high quantum yield, acridinium compounds ofthe present invention is evident in heterogeneous immunoassays for twoanalytes. For example, theophylline is a small molecule analyte and thetheophylline assay serves as an example of the competitive assays of thepresent invention. Thyroid stimulating hormone (TSH) is a macromolecularanalyte that is commonly measured by immunoassays. The TSH assay is usedas an example of the sandwich assays.

For the theophylline assay, the assay performance of twotheophylline-acridinium conjugates was compared, the conjugateNSP-DMAE-HEG-theophylline described in U.S. Pat. No. 6,664,043 and thehigh quantum yield hydrophilic conjugateNSP-2,7-(OMHEG)₂-DMAE-HEG-theophylline described in Example 15.

The assay is described in Example 18 and the structures of the twoconjugates are as follows:

The Bayer Diagnostics ACS:180® Theophylline Assay is one of a series ofcommercially marketed immunoassays manufactured by Bayer Diagnostics forapplication on the ACS:180® (Automated Chemiluminescent ImmunoassaySystem).

The assay is a competitive immunoassay which uses a chemiluminescentacridinium compound conjugate of theophylline,NSP-DMAE-HEG-theophylline, for measurement of theophylline in a sample.In the theophylline assay, two reagents were mixed with the samplecontaining the analyte theophylline to start the assay. The first assayreagent is an anti-theophylline antibody immobilized on paramagneticparticles (PMP) which binds both the analyte, which is theophylline, andthe theophylline-acridinium ester conjugates. The second assay reagentis the theophylline-acridinium ester conjugate.

Since the solid phase has a limited amount of the anti-theophyllineantibody, the theophylline analyte in the sample and thetheophylline-acridinium ester conjugate compete for binding to theantibody on the solid phase. Therefore, the amount of analyte in asample is inversely correlated to the amount of thetheophylline-acridinium ester conjugate that will bind to the solidphase in the assay.

The Bayer Diagnostics ACS:180® automatically performed the followingsteps for the theophylline Assay. First, 0.020 mL of each of fourteensamples was dispensed into a separate cuvet. A cuvet is an opticallytransparent or translucent container that holds the assay reagents andin which the assay takes place.

The fourteen samples each contained separate known amounts oftheophylline. The amounts of theophylline given as concentrations ineach of these fourteen samples were 0, 1.40, 2.10, 2.80, 4.20, 5.60,9.21, 15.6, 32.7, 68.3, 129, 288, 500, and 1000 micromolar (uM). Theamounts of theophylline given as numbers of molecules in each of thesesame fourteen samples were 0, 0.028, 0.042, 0.056, 0.084, 0.112, 0.184,0.313, 0.655, 1.37, 2.59, 5.76, 10.0, and 2.00 picomoles (10⁻¹² moles),respectively.

Next, the ACS:180® dispensed the two assay reagents together into eachcuvet and mixed the assay reagents with the sample within each cuvet.The first of the two assay reagents was 0.450 mL of solid phase, whichcontained 8.7 picomoles of anti-theophylline antibody on magneticallyseparable paramagnetic particles. The second of the two assay reagentswas 0.100 mL of theophylline-acridinium ester conjugate, which was 0.026picomole of acridinium compound conjugated to theophylline.

The assay proceeded for 7.5 minutes at 37° C. The Bayer DiagnosticsACS:180® finished the assay by magnetically separating the solid phasefrom other assay reagents, then removing fluid from the cuvet and thenwashing the solid phase in the cuvet with water. Chemiluminescence fromthe acridinium compound on the solid phase was initiated with subsequentlight emission with the sequential additions of 0.30 mL each of BayerDiagnostics ACS:180® Reagent 1 and Bayer Diagnostics ACS:180® Reagent 2.Reagent 1 was 0.1 M nitric acid and 0.5% hydrogen peroxide. Reagent 2was 0.25 M sodium hydroxide and 0.05% cetyltrimethylammonium chloride.

The Bayer Diagnostics ACS:180® measured the chemiluminescence in eachcuvet with each cuvet corresponding to a single assayed sample asrelative light units (RLUs). Normalization to percentage ofchemiluminescence measured in the absence of analyte was calculated forcomparison of the relative chemiluminescence given for each amount ofanalyte for the two theophylline-acridinium ester conjugates. The assayresults are tabulated in Table 2 below and in graphical form in FIG. 1.

The spacing between chemiluminescence values for successive amounts ofanalyte is an indicator of assay sensitivity with greater spacingequating to greater sensitivity. This is well known to practitioners inthe field. In the current assay, relative to the lower quantum yieldlabel NSP-DMAE-HEG, the high quantum yield acridinium compoundNSP-2,7-(OMHEG)₂-DMAE-HEG generated greater differentiation betweensamples containing small amounts of theophylline and no theophylline.

TABLE 2 Theophylline Assay Acridinium Compound Label NSP-DMAE-HEGNSP-2,7-(OMHEG)2-DMAE-HEG Theophylline Chemiluminescence [μM] (RLU) (%)(RLU) (%) 0 1846515 100 2493756 100 1.40 1774943 96.1 2237897 89.7 2.101806959 97.9 2115736 84.8 2.80 1790450 97.0 1959285 78.6 4.20 169864192.0 1770358 71.0 5.60 1609515 87.2 1647086 66.0 9.21 1391668 75.41523003 61.1 15.6 1119475 60.6 1056765 42.4 32.7 808738 43.8 682131 27.468.3 525335 28.5 395396 15.9 129 314966 17.1 217917 8.74 288 158037 8.56107430 4.31 500 100707 5.45 61806 2.48 1000 51284 2.78 32293 1.29

The slope of the line generated for each tracer using the BayerDiagnostics ACS:180® Theophylline Assay is an indicator of sensitivity.In the present assay, the high quantum yield acridinium compound labelNSP-2,7-(OMHEG)₂-DMAE-HEG gave enhanced slope relative to the acridiniumcompound label NSP-DMAE-HEG at all concentrations of the analytetheophylline.

Assay sensitivity is often defined as the least measurable amount ofanalyte. The least measurable amount of analyte in the currentcompetitive immunoassay is the amount of analyte corresponding to thegreatest measured chemiluminescence that is less than the difference ofthe chemiluminescence measured in the absence of analyte minus twostandard deviations of chemiluminescence measured in the absence ofanalyte. For example in competitive immunoassays where the followingrepresentations are given:

n=positive integer greater than 0.

x=the measured amount of analyte corresponding to y, where x0<x1<x2<x3<. . . <xn are successively greater measured amounts of analyte.

y=the chemiluminescence measured for an amount of analyte, representedby x, where y0>y1>y2>y3> . . . >yn are successively lesser values ofchemiluminescence, measured for x0<x1<x2<x3< . . . <xn, respectively.

x0=a zero amount of analyte or the amount of analyte equal to zero.

y0=the chemiluminescence measured for an amount of analyte equal tozero, which is x0.

s=one standard deviation of y0.

Then the sensitivity=xn for yn<y0−2s, when n=the least, positive,nonzero integer.

Using this definition, the sensitivity of the theophylline assay usingthe high quantum yield acridinium compound NSP-2,7-(OMHEG)₂-DMAE-HEG was1.4 μM. The sensitivity of the assay using the acridinium compoundNSP-DMAE-HEG was 4.2 μM. The quotient of 4.2 μM and 1.4 μM is 3.

The high quantum yield acridinium compound NSP-2,7-(OMHEG)₂-DMAE-HEGthus enhanced the sensitivity of the Bayer Diagnostics ACS:180®Theophylline Assay three-fold when compared to the acridinium compoundNSP-DMAE-HEG.

The example clearly establishes that when used as chemiluminescentimmunoassay labels the enhanced chemiluminescent light emission fromhigh quantum yield acridinium compounds enhances the sensitivity ofcompetitive immunoassays.

For the TSH assay, the assay performance of the acridinium esterNSP-DMAE-HEG-glutarate-NHS described in U.S. Pat. No. 6,664,043 wascompared with the hydrophilic, high quantum yield acridinium compoundsof the present invention, which is described in Example 19.

A comparison was made of the compounds NSP-2,7-(OMTEG)₂-DMAE-NHS,NSP-2,7-(OMTEG)₂-DMAE-HEG-glutarate-NHS, NSP-2,7-(OMHEG)₂-DMAE-AC-NHSand NSP-2,7-(OMHEG)₂-HEG-glutarate-NHS whose structures are shown belowand whose syntheses are described in Example 9 (HEG-containingcompounds) and Example 10 (TEG-containing compounds) respectively.

The Bayer Diagnostics ACS:180® TSH3 assay is one of a series ofcommercially marketed immunoassays manufactured by Bayer Diagnostics forapplication on the ACS:180®. The TSH3 assay is a sandwich immunoassaywhich uses a chemiluminescent acridinium compound conjugate of ananti-TSH antibody, for measurement of the analyte TSH (ThyroidStimulating Hormone) in a sample. In this assay there are two antibodiesone of which is labeled with acridinium ester while the other isimmobilized on paramagnetic particles (PMP).

In the assay, the two antibodies are mixed with the sample containingthe analyte TSH to start the assay. This results in the binding of boththe antibodies to the TSH analyte. As the concentration of the analyteTSH increases, a greater amount of the acridinium ester labeled antibodyis bound to the solid phase. Thus, the concentration of analyte isdirectly correlated with the amount of chemiluminescent signal observedin the assay.

The ACS:180® automatically performed the following steps for the currentTSH3 assay. First, 200 μl of each of twelve samples was dispensed intoseparate cuvets. The twelve samples each contained separate knownamounts of TSH. The amounts of TSH given as concentrations in each ofthese twelve samples were 0, 0.002, 0.004, 0.010, 0.015, 0.020, 0.025,0.030, 0.10, 1.0, 10 and 100 mIU/L. Next, the ACS:180® dispensed twoassay reagents together into each cuvet and mixed the assay reagentswith the sample within each cuvet. The first of the two assay reagentswas 0.100 mL of a solution, which contained 0.22 picomoles of anti-TSHantibody conjugated with acridinium compound.

Both the high quantum yield acridinium compounds andNSP-DMAE-HEG-glutarate were tested separately as labels conjugated tothe anti-TSH antibody. The conjugates were prepared and purified usingthe procedure described in example 16. The number of labels per antibodymolecule for NSP-DMAE-HEG-glutarate, NSP-2,7-(OMTEG)₂-DMAE,NSP-2,7-(OMTEG)₂-DMAE-HEG-glutarate, NSP-2,7-(OMHEG)₂-DMAE-AC andNSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate were 8, 8, 7, 10 and 8 respectively.

Thus, all the conjugates contained an approximately equivalent number oflabels. Therefore, any difference in assay performance can be correlateddirectly to the properties of the acridinium compound label.

The binding reaction proceeded for 2.5 minutes at 37° C. The second ofthe two assay reagents was 0.225 mL of solid phase, which was the otheranti-TSH antibody conjugated to paramagnetic particles. The assay thenproceeded for 5.0 minutes at 37° C. The assay was finished bymagnetically separating the solid phase from other assay reagents,removing fluid from the cuvet and, washing the solid phase in the cuvetwith water.

Chemiluminescence from the acridinium compound on the solid phase wasinitiated with subsequent light emission with sequential additions of0.30 mL each of Bayer Diagnostics ACS:180® Reagent 1 and BayerDiagnostics ACS:180® Reagent 2. The chemiluminescence in each cuvet wasthen measured as relative light units (RLUs) with each cuvetcorresponding to a single assayed sample. The amount of analyte iscorrelated with the number of RLUs measured by the Bayer DiagnosticsACS:180®. The greater the amount of the analyte TSH in a sample, thegreater the amount of RLUs that are measured.

In the current assay, the high quantum yield acridinium compound labelsNSP-2,7-(OMTEG)₂-DMAE, NSP-2,7-(OMTEG)₂-DMAE-HEG-glutarate,NSP-2,7-(OMHEG)₂-DMAE-AC and NSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate gaveenhanced chemiluminescence for all amounts of analyte relative to theacridinium compound label NSP-DMAE-HEG-glutarate. The results of theassay are tabulated in Tables 3-5 and plotted graphically in FIG. 2.

TABLE 3 TSH Assay using Acridinium Compounds as Labels NSP-DMAE-HEG-NSP-2,7- NSP-2,7-(OMHEG)₂- NSP-2,7-(OMHEG)₂- NSP-2,7-(OMHEG)₂- TSHglutarate (OMTEG)₂-DMAE DMAE-HEG-glutarate DMAE-AC DMAE-HEG-glutarate[mI.U./L] Chemiluminescence (RLU) 0 12504 13492 20114 11871 12343 0.00212831 14514 20185 12567 13973 0.004 13127 15567 22650 13084 14152 0.0113680 17442 24519 13418 15493 0.015 14161 21150 25387 15268 17577 0.0215106 21628 27810 16831 19542 0.025 15558 22920 29258 18547 21139 0.0315750 23840 30872 20170 22404 0.1 20998 50110 66057 46578 58692 1 62108306958 352706 286667 283798 10 553358 1916772 3147811 1867356 1999399100 4350612 16861210 21937230 15763393 17584945

Noise is the portion of chemiluminescence in a sandwich immunoassay of asample which is due to the non-specific binding of the labeled antibodyto the solid phase and which is measured in samples that contain noanalyte. Signal is the portion of the chemiluminescence due to thespecific binding of the labeled antibody to the solid phase when analyteis present in the sample. The total chemiluminescence measured in thecurrent TSH assay for samples that do contain analyte is the sum ofsignal plus noise, where signal is calculated as the difference of thetotal chemiluminescence minus the noise.

For the current assay, sensitivity is defined as the least measurableamount of analyte which corresponds to the least measuredchemiluminescence that is greater than the sum of the noise plustwo-standard deviations of the noise. In the current assay signal andnoise were determined for each tested antibody-acridinium esterconjugate. The ratio of the signal divided by the noise in a sandwichimmunoassay for a particular amount of analyte is an indicator ofsandwich immunoassay sensitivity. The greater the signal to noise ratiofor a particular amount of analyte in a sandwich immunoassay, the moredistant is the corresponding signal from the noise and the better ableis the assay to measure the difference between the signal and the noise.

In the current assay, the high quantum yield acridinium compound labelsNSP-2,7-(OMTEG)₂-DMAE, NSP-2,7-(OMTEG)₂-DMAE-HEG-glutarate,NSP-2,7-(OMHEG)₂-DMAE-AC and NSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate gaveenhanced signal to noise ratios for the analyte relative to the labelNSP-DMAE-HEG-glutarate for all concentrations of the analyte. Thegreater signal to noise ratios generated by the high quantum yieldacridinium compound labels for both the high amounts and particularlythe low amounts of TSH, relative to NSP-DMAE-HEG-glutarate, indicate anenhancement of sensitivity for the TSH assay.

When the results of Table 3 are plotted in graphical form in FIG. 2,then the slope of the line generated for each antibody-acridinium esterconjugate is also an indicator of sensitivity. The greater the slope ofthe line, the more distant is the signal for a particular amount ofanalyte from the noise and the assay is better able to measure thedifference between the signal and the noise. In the current assay, thehigh quantum yield acridinium compound labels NSP-2,7-(OMTEG)₂-DMAE,NSP-2,7-(OMTEG)₂-DMAE-HEG-glutarate, NSP-2,7-(OMHEG)₂-DMAE-AC andNSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate gave enhanced slopes relative toNSP-DMAE-HEG-glutarate as is evident from FIG. 2. The slopes of thelines in FIG. 2 are tabulated in Table 4.

TABLE 4 TSH Assay Slopes using Acridinium Compounds as Labels NSP-2,7-NSP- (OMTEG)₂- NSP-2,7- DMAE- NSP-2,7- DMAE- NSP-2,7- (OMHEG)₂- HEG-(OMTEG)₂- HEG- (OMHEG)₂- DMAE-HEG- glutarate DMAE glutarate DMAE-ACglutrate Low TSH Slope from 0 to 0.03 1.1E+05 3.4E+05 3.6E+05 2.8E+053.4E+05 (RLU/[mI.U./L]) mI.U./L TSH High TSH Slope from 1 to 100 mI.U./L4.3E+04 1.7E+05 2.2E+05 1.6E+05 1.7E+05 (RLU./(mI.U./L]) TSH

Assay sensitivity is often defined as the least measurable amount ofanalyte. In the current sandwich immunoassay the least measurablenon-zero amount of analyte is the amount of analyte corresponding to theleast measured chemiluminescence that is greater than the sum of thenoise plus two-standard deviations of the noise. For example in sandwichimmunoassays where the following representations are given:

n=positive integer greater than 0.

x=the measured amount of analyte corresponding to y, where x0<x1<x2<x3<. . . <xn are successively greater measured amounts of analyte.

y=the chemiluminescence measured for an amount of analyte, representedby x, where y0<y1<y2<y3< . . . <yn are successively greater values ofchemiluminescence, measured for x0<x1<x2<x3< . . . <xn, respectively.

x0=a zero amount of analyte or the amount of analyte equal to zero.

y0=the chemiluminescence measured for an amount of analyte equal tozero, which is x0.

s=one standard deviation of y0.

Then the sensitivity=xn for yn>y0+2s, when n=the least, positive,nonzero integer.

The assay sensitivity in the current assay using the high quantum yieldacridinium compound labels were enhanced as shown in Table 5 with thelower numbers indicative of a more sensitive assay. For example, usingthe hydrophilic, high quantum yield labelNSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate in the assay affords an assaysensitivity that is >7-fold more sensitive (0.015/0.002) when comparedto the label NSP-DMAE-HEG-glutarate. The example clearly demonstratesthat when used as labels, hydrophilic high quantum yield acridiniumcompounds enhances the sensitivity of sandwich immunoassays.

TABLE 5 TSH Assay Sensitivity using Acridinium Compounds as LabelsNSP-2,7- NSP-2,7- (OMTEG)₂- NSP-2,7- (OMHEG)₂- NSP-DMAE-HEG- NSP-2,7-DMAE-HEG- (OMHEG)₂-DMAE- DMAE-HEG- glutarate (OMTEG)₂-DMAE glutarate ACglutarate TSH [mI.U./L] 0.015 0.004 0.004 0.010 0.002

The enhanced sensitivity in the theophylline assay and TSH assay usingthe high quantum yield acridinium compounds of the present inventionillustrates, in general, the utility of these compounds in enhancingassay sensitivity of a variety of assays. There are numerous methods ofdesigning immunoassays and nucleic acid assays, which are well known inthe art. Regardless of the assay design, if an assay for an analyterelies on the generation of a chemiluminescent signal for measurement ofthe concentration of that analyte, then the acridinium compounds of thepresent invention, because of their high quantum yield and hydrophilicnature, will enable a more sensitive measurement of concentration ofthat analyte.

The chemiluminescent acridinium compounds suitable for use in thepresent invention have the following structure:

wherein,

R₁ is selected from an alkyl, alkenyl, alkynyl or aralkyl containing upto 20 heteroatoms; preferably a methyl, a sulfopropyl, or a sulfobutylgroup;

R₂ and R₃ can be the same or different, and are selected from groupscomprising hydrogen, halides or R where R is selected from an alkyl,alkenyl, alkynyl, aryl, or aralkyl containing up to 20 heteroatoms atpositions other than C-2 and C-7;

X is oxygen or nitrogen;

when X is oxygen, Z is omitted and Y is a substituted aryl moiety of theformula:

where R₄ and R₈ can be the same or different and are selected fromhydrogen, alkyl, alkenyl, alkynyl, alkoxyl (—OR), alkylthiol (—SR), or—NR_(a)R_(b) groups where R_(a) and R_(b) can be the same or different,and are selected from alkyl, alkenyl, alkynyl, aryl, or aralkylcontaining up to 20 heteroatoms, R₄ and R₈ are preferably methyl;

R₅ and R₇ are the same or different and are hydrogen or the same as R asdefined above;R₆═—R₉—R₁₀,

where R₉ is not required and is selected from branched orstraight-chained alkyl, substituted or unsubstituted aryl or aralkylcontaining up to 20 heteroatoms, and

R₁₀ is a leaving group or an electrophilic functional group attachedwith a leaving group selected from the group consisting of:

a halide or —COOH;

R₅ and R₆, and R₆ and R₇ are interchangeable.

Alternatively, when X is oxygen, Z is omitted and Y is —N═C(R₁₁R₁₂)where R₁₁ and R₁₂ are the same as R₆ defined above and can be the sameor different;

when X is nitrogen, Y is the same as R₆ defined above, Z is —SO₂—Y′, andY′ is a substituted or unsubstituted aryl group or branched or straightchain;

W₁ and W₂ are the same or different and are electron-donating groupscomprising —OR, —OH, —SR, —SR, —NH₂, —NR′R″; wherein R, R′ and R″ can bethe same or different, and are selected from the group consisting ofalkyl, alkenyl, alkynyl, aryl, and aralkyl containing up to 20heteroatoms; preferably W₁ and W₂ can be the same or different and areselected from —OMe, —OCH₂CH₂CH₂SO₃ ⁻ and —O(CH₂CH₂O)_(n)—CH₂—CH₂—OMe,wherein Me represents a methyl group and n=0-5.

A⁻ is a counter ion which is introduced to pair with the quaternarynitrogen of said acridinium nucleus, and is selected from the groupconsisting of CH₃SO₄ ⁻, FSO₃ ⁻, CF₃SO₄ ⁻, C₄F₉SO₄ ⁻, CH₃C₆H₄SO₃ ⁻,halide, CF₃COO⁻, CH₃COO⁻, and NO₃ ⁻.

More specifically, the acridinium compound can be an acridinium ester ofthe following structure:

where R₁₃ is selected from —OH, —O—N-succinimidyl,—NH—(CH₂)₅—C(O)—O—N-succinimidyl,—NH—(C₂H₄O)_(n)—C₂H₄NH—C(O)—(CH₂)₃—C(O)—O—N-succinimidyl, wherein n=0 to5, or —NH—R—NHR, and where R₁, A⁻, and R are as described previously.

The acridinium compound can also be an acridinium ester of the followingstructure:

where R₁₃, R₁ and A⁻ are defined above, and X⁺ is a positively chargedcounterion to pair with the sulfonate moiety and can include H⁺, Na⁺,K⁺, or NH₄ ⁺.

The acridinium compound can also be an acridinium ester of the followingstructure:

where R₁₃, R₁ and A⁻ are defined previously.

The acridinium compound can also be an acridinium ester of the followingstructure:

where R₁₃, R₁, A⁻ and X+ are defined previously.

The acridinium compound can also be an acridinium ester of the followingstructure:

where R₁₃, R₁ and A⁻ are defined previously, and n=0 to 5.

The acridinium compound can also be an acridinium ester of the followingstructure:

where R₁₃, R₁ and A⁻ and n are described previously.

The acridinium compound can also be an acridinium ester of the followingstructure:

The acridinium compound can also be an acridinium ester of the followingstructure:

where n and R₁₃ are as described previously.

The acridinium compound can also be an acridinium ester of the followingstructure:

where n and R₁₃ are as described previously.

The acridinium compound can also be an acridinium sulfonamide of thefollowing structure:

The acridinium compound can also be an acridinium sulfonamide of thefollowing structure, where n=0 to 5.

Example 1 Synthesis of 2′,6′-dimethyl-4′-carboxyphenyl2,7-dimethoxy-10-N-sulfopropyl-acridinium-9-carboxylate[NSP-2,7-(OMe)₂-DMAE] and its N-succinimidyl ester[NSP-2,7-(OMe)₂-DMAE-NHS] (a) Synthesis of2′,6′-dimethyl-4′-benzyloxycarbonylphenyl2,7-dimethoxy-acridine-9-carboxylate

2,7-Dimethoxy acridine-9-carboxylic acid (U.S. Pat. No. 5,521,103) (0.5g, 0.00177 mol) in pyridine (˜25 mL) was treated with tosyl chloride(0.674 g, 2 equivalents). After 10 minutes of stirring,4-benzyloxycarbonyl-2,6-dimethylphenol (0.453 g, 1 equivalent) was addedand the resulting mixture was stirred at room temperature under anitrogen atmosphere. After 1-2 hours, an additional 2 equivalents oftoluenesulfonyl chloride was added along with 0.5 equivalent of thephenol and 10-15 mL pyridine. The reaction was stirred at roomtemperature under nitrogen atmosphere for 48 hours. The solvent was thenremoved under reduced pressure and the residue was dissolved in 50 mLchloroform. This solution was washed with 2% aqueous ammonium chlorideand 2% aqueous sodium bicarbonate. The chloroform extract was then driedover magnesium sulfate, filtered and evaporated to dryness. The crudeproduct was purified by preparative TLC using 5% ethyl acetate inchloroform. Yield=0.663 g (72%). MALDI-TOF MS 524.3 obs. (521.6 calc.).

(b) Synthesis of 2′,6′-dimethyl-4′-carboxyphenyl2,7-dimethoxy-10-N-sulfopropyl-acridinium-9-carboxylate

The acridine ester from above (20 mg, 38.4 umoles), 1,3-propane sulfone(0.28 g, 2.29 mmoles) and sodium bicarbonate (32 mg, 384 umoles) weremixed in a 10 mL round bottom flask and heated in an oil-bath at 120° C.under a nitrogen atmosphere. After 4 hours, the reaction was cooled toroom temperature and diluted with ethyl acetate (10 mL). The suspensionwas sonicated until the gummy solid was dispersed into the solvent togive a reddish-yellow precipitate. This precipitate was collected byfiltration and rinsed with ethyl acetate. It was then dissolved inmethanol and filtered. HPLC analysis of the filtrate using a C₁₈ column(Phenomenex 4.6 mm×30 cm) and a 30 minute gradient of 10-70% MeCN inwater (each solvent with 0.05% trifluoroacetic acid) showed producteluting at 23 minutes with ˜10% starting material eluting at 31 minutes.The methanol solution was evaporated to dryness to give 42 mg of crudeproduct which was stirred in 2 mL of 30% HBr/AcOH at room temperature.Ether (30 mL) was added after 6 hours to precipitate the product, whichwas collected by filtration and rinsed with ether. The product wasdissolved in methanol (40 mL) and analyzed by HPLC as described above.The product was found to elute at 15.9 minutes with no startingmaterial. Evaporation of the methanol filtrate afforded an oily solid,which was re-dissolved in methanol (2-3 mL) and diluted with ethylacetate (20 mL). Evaporation of the solvent yielded a yellow solid.Yield=34 mg. A portion of this material was dissolved in DMF (2-3 mL)and purified by preparative HPLC using a 30 mm×30 cm C₁₈ column. TheHPLC fraction, containing product was frozen at −80° C. and, lyophilizedto dryness to give a bright yellow powder. MALDI-TOF MS 555.7 obs.(553.6 calc.).

(c) Synthesis of 2′,6′-dimethyl-4′-N-succinimidyloxycarbonyphenyl2,7-dimethoxy-10-N-sulfopropyl-acridinium-9-carboxylate

Crude NSP-2,7-(OMe)₂-DMAE (34 mg, 61.5 umoles,) in DMF (3 mL) wastreated with diisopropylethylamine (13 uL, 1.2 equivalents) and TSTU(18.5 mg, 1 equivalent). After stirring for 15 minutes, HPLC analysis asdescribed in step (b) r, indicated complete conversion to producteluting at 17.8 minutes. A portion of the above reaction mixture waspurified by preparative HPLC as described in step (b). The HPLCfraction, containing product was frozen at 80° C. and lyophilized todryness to give a bright yellow powder. MALDI-TOF MS 653.2 obs. (650.7calc.).

The following reactions describe the synthesis of NSP-2,7-(OMe)₂-DMAEand its NHS ester.

Example 2 Synthesis of 2′,6′-dimethyl-4′-carboxyphenyl 2-methoxy-10-Nsulfopropyl-acridinium-9-carboxylate (NSP-2-OMe-DMAE) (a) Synthesis ofN-(4-methoxyphenyl)isatin

A solution of isatin (2.5 g, 0.017 mol) in anhydrous DMF (50 mL) wascooled to 0° C. in an ice-bath and treated with sodium hydride (0.5 g,1.2 equivalents). A purple solution was formed which was stirred at 0°C. for 30 minutes and then warmed to room temperature. To this solutionwas added 4-bromoanisole (2.13 mL, 1 equivalent) followed by copperiodide (6.46 g, 2 equivalents). The reaction was heated in an oil-bathat 145° C. for 6-7 hours. The reaction was then cooled to roomtemperature, diluted with an equal volume of ethyl acetate and filtered.The filtrate was evaporated to dryness. The crude product, which wasobtained as a dark brown solid was used as such for the next reaction.

(b) Synthesis of 2-methoxy-acridine-9-carboxylic acid

The isatin from step (a) was refluxed with 150 mL of 10% potassiumhydroxide under a nitrogen atmosphere. After 4 hours, the reaction wascooled to room temperature and filtered. The filtrate was diluted withwater (150 mL) and ice. This solution was then acidified withconcentrated HCl. A yellow precipitate separated out which was collectedby filtration and rinsed with cold water and ether. After air drying,the precipitate was transferred to a round-bottom flask with methanoland evaporated to dryness. The resulting residue was evaporated todryness twice from toluene and was obtained as a yellowish brown powder.Yield=1.15 g (26%).

(c) Synthesis of 2′,6′-dimethyl-4′-benzyloxycarbonylphenyl2-methoxy-acridine-9-carboxylate

2-Methoxy acridine-9-carboxylic acid (0.8 g, 0.0032 mol) in pyridine (30mL) was cooled in an ice-bath under a nitrogen atmosphere and treatedwith tosyl chloride (1.32 g, 2 equivalents) followed by4-benzyloxycarbonyl-2,6-dimethylphenol (0.81 g). The reaction was warmedto room temperature and stirred for 24 hours. The solvent was thenremoved under reduced pressure and the residue was suspended in 100 mLtoluene and evaporated to dryness. The resulting residue was dissolvedin chloroform (10 mL) and purified by flash chromatography on silica gelusing 5% ethyl acetate, 25% chloroform 70% hexanes. Yield=0.84 g (54%),bright yellow powder. MALDI-TOF MS 492.8 obs. (491.5 calc.).

(d) Synthesis of2′,6′-dimethyl-4′-carboxyphenyl-2-methoxy-10-N-sulfopropyl-acridinium-9-carboxylate

This compound was synthesized from the acridine ester by the methoddescribed in Example 1, section (b) for NSP-2,7-(OMe)₂-DMAE.

(e) Synthesis of 2′,6′-dimethyl-4′-N-succinimidyloxycarbonyphenyl2-methoxy-10-N-sulfopropyl-acridinium-9-carboxylate

NSP-2-OMe-DMAE (23 mg, 0.044 mmol) in DMF (1.5 mL) was treated withN-hydroxysuccinimide (25 mg, 5 equivalents) and diisopropyl carbodiimide(68 uL, 10 equivalents). The reaction was stirred vigorously at roomtemperature. After 36 hours, HPLC analysis, as described earlier inExample 1, section (b), showed complete conversion to product eluting at16.7 minutes with no starting material at 14.7 minutes. The product waspurified by preparative HPLC using a C₁₈, 20 mm×30 cm column. The HPLCfraction, containing product was frozen at −80° C. and lyophilized todryness to give a bright yellow powder. Yield=23 mg (84%).

The following reactions describe the synthesis of NSP-2-OMe-DMAE

Example 3 Synthesis of 2′,6′-dimethyl-4′-carboxyphenyl3-methoxy-10-N-sulfopropyl-acridinium-9-carboxylate [NSP-3-OMe-DMAE]

This compound was made from 3-bromoanisole, isatin and4-benzyloxycarbonyl-2,6-dimethylphenol using the same proceduresdescribed above for the 2-methoxy analog in Example 2.

The following reactions describe the synthesis of NSP-3-OMe-DMAE.

Example 4 Synthesis of 2′,6′-dimethyl-4′-carboxyphenyl4-methoxy-10-N-sulfopropyl-acridinium-9-carboxylate [NSP4-OMe-DMAE]

This compound was made from 2-bromoanisole, isatin and4-benzyloxycarbonyl-2,6-dimethylphenol using the same proceduresdescribed above for the 2-methoxy analog in Example 2. The followingreactions describe the synthesis of NSP4-OMe-DMAE.

Example 5 Synthesis of 2′,6′-dimethyl-4′-carboxyphenyl2,4-dimethoxy-10-N-sulfopropyl-acridinium-9-carboxylate[NSP-2.A(OMe)₂-DMAE]

This compound was made from 2,4-dibromoanisole, isatin and4-benzyloxycarbonyl-2,6-dimethylphenol using the same proceduresdescribed above for the 2-methoxy analog in Example 2. The followingreactions describe the synthesis of

Example 6 Synthesis of 2′,6′-dimethyl-4′-carboxyphenyl2,5-dimethoxy-10-sulfopropyl-acridinium-9-carboxylate[NSP-2,5-(OMe)₂-DMAE] (a) Synthesis ofN-(2-methoxyphenyl)-5-methoxyisatin

5-Methoxyisatin (2 g, 0.0113 mol) in anhydrous DMF (15 mL) was cooled inan ice-bath under a nitrogen atmosphere and treated with sodium hydride(0.54 g, 1.2 equivalents, 60% dispersion in mineral oil). The reactionwas stirred at 0° C. for 0.5 hour and then 2-bromoanisole (1.4 mL, 1equivalent) was added along with copper iodide (4.3 g, 2 equivalents).The reaction was heated in an oil-bath at 140° C. for 16 hours. Thereaction was then cooled to room temperature and filtered. The filtratewas evaporated to dryness. The dark brown oily solid was used directlyfor the next reaction.

(b) Synthesis of 2,5-dimethoxy acridine-9-carboxylic acid

The crude isatin derivative from step (a) was refluxed in 60 mL of 10%potassium hydroxide in water for 4 hours. The reaction was then cooledto room temperature and filtered. The filtrate was acidified with amixture of concentrated hydrochloric acid and ice until a darkprecipitate separated out. The product was collected by filtration anddried under vacuum. Yield=0.32 g (10%).

(c) Synthesis of2′,6′-dimethyl-4′-benzyloxycarbonylpheny-2,5-dimethoxy-acridine-9-carboxylate

This compound was synthesized from 2,5-dimethoxy acridine-9-carboxylicacid and 4-benzyloxycarbonyl-2,6-dimethylphenol using the proceduredescribed for the synthesis of the 2,7-dimethoxy analog in Example 1.

(d) Synthesis of 2′,6′-dimethyl-4′-carboxyphenyl2,5-dimethoxy-10-N-sulfopropyl-acridinium-9-carboxylate[NSP-2,5-(OMe)₂-DMAE]

This compound was synthesized by reaction of the acridine ester with1,3-propane sultone using the procedure described for the synthesis ofthe NSP-2,7-(OMe)₂-DMAE in Example 1, section (b).

The following reactions describe the synthesis of NSP-2,5-(OMe)₂-DMAE.

Example 7 Synthesis of 2′,6′-dimethyl-4′-carboxyphenyl2,4,7-trimethoxy-10-N-sulfopropyl-acridinium-9-carboxylate[NSP-2,4,7-(OMe)₃-DMAE]

This compound was synthesized from 5-methoxyisatin, 2,4-dibromoanisoleand 2,6-dimethyl-4-carboxybenzylphenol using procedures described inExample 6.

The following reactions describe the synthesis of NSP-2,4,7-(OMe)₃-DMAE.

Example 8 Synthesis of 2′,6′-dimethyl-4′-carboxyphenyl2,7-bis(O-sulfopropyl)-10-N-sulfopropyl-acridinium-9-carboxylate[NSP-2.7-(OSP)₂-DMAE] and its N-succinimidyl ester[NSP-2,7-(OSP)₂-DMAE-NHS] (a) Synthesis of2′,6′-dimethyl-4′-methoxycarbonylphenyl2,7-dihydroxy-acridine-9-carboxylate

A solution of2′,6′-dimethyl-4′-benzyloxycarbonylphenyl-2,7-dimethoxy-acridine-9-carboxylate(52 mg, 100 umoles) in dichloromethane (5 mL) was cooled in an ice-bathunder a nitrogen atmosphere and treated with a 1M solution of borontribromide in dichloromethane (4 mL). The reaction was warmed to roomtemperature and stirred for 34 hours. The reaction was then cooled againin an ice-bath and methanol (10 mL) was added carefully. The reactionwas then warmed to room temperature and stirred for 16 hours. After theindicated reaction time, solid sodium bicarbonate was added to thereaction till neutral by pH paper. The whole reaction mixture was thenevaporated to dryness. The residue was partitioned between ethyl acetate(30 mL) and water (50 mL). The ethyl acetate layer was dried overanhydrous magnesium sulfate, filtered and evaporated to dryness to givea yellow powder. Yield=30 mg (75%). MALDI-TOF MS 419.4 obs. (420.5calc.).

(b) Synthesis of 2′,6′-dimethyl-4′-carboxyphenyl2,7-bis(O-sulfopropyl)-10-N-sulfopropyl acridinium-9-carboxylate

The 2,7-dihydroxy acridine derivative from section (a) (30 mg, 71.6umoles) was mixed with 1,3-propane sultone (0.875 g, 100 equivalents)and sodium bicarbonate (120 mg, 20 equivalents). The mixture was heatedin an oil-bath at 140° C. with vigorous stirring. After one hour,additional 100 equivalents of 1,3-propane sultone and 20 equivalents ofsodium bicarbonate were added and the reaction was continued. After anadditional hour, the reaction mixture was treated with more 1,3-propanesultone (100 equivalents) and sodium bicarbonate (20 equivalents). Thereaction was continued for an hour and then cooled to room temperature.Ethyl acetate (20 mL) was added and the reaction mixture was sonicatedbriefly (15 minutes) to disperse the gum into a yellow precipitate. Thisprecipitate was collected by filtration and rinsed with ethyl acetate.This solid and then transferred with the help of methanol to around-bottom flask and was then evaporated to dryness. The resultingresidue was suspended in 20 mL of IN hydrochloric acid and was refluxedfor 3 hours. HPLC analysis as described in Example 1, section (b),indicated product eluting at 11 minutes along with some by-products. Thereaction mixture was concentrated to a small volume and was purified bypreparative HPLC using a C₁₈, 20 mm×30 cm column. The HPLC fractioncontaining the product was frozen at −80° C. and lyophilized to drynessto yield a bright yellow powder. Yield=12.5 mg (23%). 772.7 obs. (769.8calc.).

(c) Synthesis of 2′,6′-dimethyl-4′-N-succinimidyloxycarbonylphenyl2,7-bis(O-sulfopropyl)-10-N-sulfopropyl acridinium-9-carboxylate

NSP-2,7-(OSP)₂-DMAE (3 mg, 3.9 umoles) was dissolved in anhydrous DMN (1mL) and treated with diisopropylethylamine (1.4 uL) and TSTU (1.8 mg,1.2 equivalents). The reaction was stirred at room temperature. After 40minutes, additional diisopropylethylamine and TSTU was added and thereaction was continued for 30 minutes. HPLC analysis using a C₁₈ columnfrom Phenomenex, 4.6 mm×30 cm, and a 40 minute gradient of 10%→40% MeCNin water, each with 0.05% trifluoroacetic acid, at a flow rate of 1mL/min showed complete conversion to product eluting at 19 minutes withso starting material at 17 minutes. The product was purified bypreparative HPLC using a C₁₈, 20 mm×30 cm column. The product fraction,containing product was frozen at −80° C. and lyophilized to dryness.Yield=2 mg (59%). MALDI-TOF MS 868.2 obs. (866.9 calc.).

The following reactions describe the synthesis of NSP-2,7-(OSP)₂-DMAEand its NHS ester.

Example 9 Synthesis of 2′,6′-dimethyl-4′-carboxyphenyl2,7-bis[O-methoxyhexa(ethylene)glycol]-10-N-sulfopropyl-acridinium-9-carboxylate[NSP-2,7-(OMHEG)₂-DMAE]: 2′,6′-dimethyl-4′-carboxyphenyl2,7-bis[O-methoxyhexa(ethylene)glycol]-10-N-methyl-acridinium-9-carboxylate[2,7-(OMHEG)₂-DMAE]: their N-succinimidyl esters [2,7-(OMHEG)₂-DMAE-NHS]and [NSP-2,7-(OMHEG)₂-DMAE-NHS]:2′6′-dimethyl-4′-N-succinimidyloxy-glutarylamidohexa(ethylene)glycolamidocarbonylphenyl-2,7-bis[O-methoxy-hexa(ethylene)glycol]-10-sulfopropyl-acridinium-9-carboxylate[NSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate-NHS] and2′,6′-dimethyl-4′-N-succinimidyl-oxycaproylamidocarbonylphenyl-2,7-bis[O-methoxy(hexa)ethyleneglycol]-10-N-sulfopropyl-acridinium-9-carboxylate[NSP-2,7-(OMHEG)₂-DMAE-AC-NHS] (a) Synthesis of2′,6′-dimethyl-4′-methoxycarbonylphenyl-2,7-bis[O-methoxyhexa(ethylene)glycol]-acridine-9-carboxylate

A solution of 2′,6′-dimethyl-4′-methoxycarbonylphenyl-2,7-dihydroxyacridine-9-carboxylate (100 mg, 0.24 mmol) in anhydrous THF (25 mL) wascooled in an ice bath under a nitrogen atmosphere and treated withsodium hydride (60% dispersion, 25 mg, 2.5 equivalents). After stirringfor 30 minutes in the ice bath, methoxy hexa(ethylene) glycol tosylate(Yamashita et al. J. Am Chem. Soc. 1995, 117, 6249-6253, 195 mg, 3equivalents) was added. The resulting reaction was refluxed undernitrogen for 4 hours. The reaction was then cooled to room temperatureand quenched with ethyl acetate and methanol and was then evaporated todryness. The residue was partitioned between chloroform (30 mL) andsaturated sodium chloride solution (30 mL). The chloroform layer wasseparated and the aqueous layer was extracted two more times withchloroform (2×10 mL). The combined organic layer was dried overmagnesium sulfate, filtered and evaporated to dryness to give an oilsolid. Yield=0.242 g (quant.). HPLC analysis using a C₁₈ column fromPhenomenex, 4.6 mm×30 cm, and a 30 minute gradient of 10%→70% MeCN inwater, each with 0.05% trifluoroacetic acid, at a flow rate of 1 mL/minshowed complete conversion to product eluting at 24 minutes. Thismaterial was used as such for subsequent transformations.

(b) Synthesis of2′,6′-dimethyl-4′-carboxyphenyl-2,7-bis[O-methoxyhexa(ethylene)glycol]-10-N-methyl-acridinium-9-carboxylate

The acridine ester from step (a) (45 mg, 46 umoles) was dissolved inanhydrous dichloromethane (5 mL) and was treated with sodium bicarbonate(20 mg, 5 equivalents) and methyl trifluoromethanesulfonate (50 uL, 10equivalents). The reaction was stirred at room temperature for 16 hours.HPLC analysis as described in section (a) indicated product eluting at21 minutes. The reaction was filtered through glass wool to removesodium bicarbonate and the filtrate was evaporated to dryness. Theresidue was suspended in 10 mL of 1N HCl and the resulting reaction wasrefluxed under a nitrogen atmosphere for 2 hours. HPLC analysis showedclean hydrolysis of the methyl ester with the product eluting at 19minutes. The reaction mixture was then concentrated to a small volume(3-4 mL) by rotary evaporation and the product was purified bypreparative HPLC using a YMC, C₁₈, 30×300 mm column and the gradientdescribed earlier. The HPLC fraction, containing product was frozen at−80° C. and lyophilized to dryness. Yield=18.2 mg (oil), 40%.

(c) Synthesis of2′,6′-dimethyl-4′-carboxyphenyl-2,7-bis[O-methoxyhexa(ethylene)glycol]-10-N-sulfopropyl-acridinium-9-carboxylate

The acridine ester from step (a) (0.1 g, 0.102 mmol) was mixed with1,3-propane sultone (1.25 g, ˜100 equivalents) and sodium bicarbonate(170 mg, 20 equivalents). The mixture was heated in an oil-bath at 130°C. under a nitrogen atmosphere. After 2-3 hours, the reaction was cooledto room temperature and diluted with ethyl acetate (˜10 mL). The mixturewas sonicated briefly to disperse the gum into a powder, which wascollected by filtration. The product was suspended in 10 mL of 1 N HCland refluxed under a nitrogen atmosphere for 2 hours. It was then cooledto room temperature and analyzed by HPLC as described in section (a)which indicated product eluting at 17 minutes. The product was purifiedby preparative HPLC as described above and the HPLC fraction was frozenat −80° C. and lyophilized to dryness. Yield ˜10 mg (10%).

(d) Synthesis of2′,6′-dimethyl-4′-N-succinimidyloxycarbonyl-phenyl-2,7-bis[O-methoxyhexa(ethylene)glycol]-10-N-methyl-acridinium-9-carboxylate

The carboxylic acid2′,6′-dimethyl-4′-carboxyphenyl-2,7-bis[O-methoxyhexa(ethylene)glycol]-10-N-methyl-acridinium-9-carboxylate(18 mg, 18.7 umoles) in anhydrous DMF (2 mL) was treated withdiisopropylethylamine (5 uL, 1.5 equivalents) and TSTU (7 mg, 1.2equivalents). The reaction was stirred at room temperature. After 1 hourHPLC analysis using a C₁₈ column from Phenomenex, 4.6 mm×30 cm, and a 30minute gradient of 10%→70% MeCN in water, each with 0.05%trifluoroacetic acid, at a flow rate of 1 mL/min showed completeconversion to product eluting at Rt=20 minutes. The product was purifiedusing a YMC, C₁₈ 20×300 mm column. The HPLC fraction, containing productwas frozen at −80° C. and lyophilized to dryness. Yield=14.4 mg (72%).MALDI-TOF MS 1073.1 obs. (1071.2 calc.).

(e) Synthesis of2′,6′-dimethyl-4′-N-succinimidyloxycarbonylphenyl-2,7-bis[O-methoxyhexa(ethylene)glycol]-10-N-sulfopropyl-acridinium-9-carboxylate

A solution of2′,6′-dimethyl-4′-carboxyphenyl-2,7-bis[O-methoxyhexa(ethylene)glycol]-10-N-sulfopropyl-acridinium-9-carboxylate(9.3 mg, 8.6 umoles) in anhydrous DMF (2 mL) was treated withdiisopropylethylamine (2.3 uL, 1.5 equivalents) and TSTU (3 mg, 1.2equivalents). The reaction was stirred at room temperature. After 1hour, HPLC analysis as described in section (d) showed conversion toproduct eluting at 18.3 minutes. The product was purified by preparativeHPLC as described above and the HPLC fraction was frozen at −80° C. andlyophilized to dryness. Yield=6.7 mg (66%). MALDI-TOF MS 1181.1 obs.(1179.3 calc.).

(f) Synthesis of2′,6′-dimethyl-4′-aminohexa(ethylene)glycolamidocarbonylphenyl-2,7-bis[O-methoxyhexa(ethylene)glycol]-10-N-sulfopropyl-acridinium-9-carboxylate

A solution of2′,6′-dimethyl-4′-carboxyphenyl-2,7-bis[O-methoxyhexa(ethylene)glycol]-10-N-sulfopropyl-acridinium-9-carboxylate(20 mg, 18.6 umoles) in DMF (2 mL) was treated withdiisopropylethylamine (8 uL, 5 equivalents) and TSTU (14 mg, 5equivalents). The reaction was stirred at room temperature. After 30minutes, HPLC analysis performed as described in section (d) indicatedcomplete conversion to the NHS ester eluting at 18.5 minutes. This DMFsolution was added in 0.1 mL portions to a solution of diaminohexa(ethylene) glycol (U.S. Pat. No. 6,664,043, 56 mg, 5 equivalents) inDMF (1 mL). The reaction was stirred at room temperature. After 30minutes, HPLC analysis performed as described in section (d) indicatedcomplete conversion to product eluting at 16.2 minutes. The product waspurified by preparative HPLC using a YMC, C₁₈, 20×300 mm column. TheHPLC fraction containing product was evaporated to dryness. Yield 16 mg(62%).

(g)2′,6′-dimethyl-4′-N-succinimidyloxyglutarylamidohexa(ethylene)glycol-amidocarbonylphenyl-2,7-bis[O-methoxyhexa(ethylene)glycol]-10-sulfopropyl-acridinium-9-carboxylate[NSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate-NHS]

The product from section (f) (16 mg, 11 umoles) was dissolved inmethanol (1 mL) and treated with diisopropylethylamine (9.6 uL, 5equivalents) and glutaric anhydride (6 mg, 5 equivalents). The reactionwas stirred at room temperature. After 30 minutes, HPLC analysisperformed as described in section (d) indicated complete conversion toproduct eluting at 17 minutes. The solvent was then removed underreduced pressure and the residue was suspended in toluene (2 mL) andevaporated to dryness. The crude product was dissolved in DMF (1 m) andtreated with diisopropylethylamine (19.2 uL, 10 equivalents) and TSTU(33 mg, 10 equivalents). The reaction was stirred at room temperature.After 30 minutes, HPLC analysis indicated>80% conversion to producteluting at 18 minutes. The product was purified by preparative HPLC asdescribed above. The HPLC fraction containing product was frozen at −80°C. and lyophilized to dryness. Yield=10.7 mg (63%); MALDI-TOF MS 1557.8obs. (1556.8 calc.).

(h) Synthesis of2′,6′-dimethyl-4′-carboxycaproylamidocarbonylphenyl-2,7-bis[O-methoxy(hexa)ethyleneglycol]-10-N-sulfopropyl-acridinium-9-carboxylate[NSP-2,7-(OMHEG)₂-DMAE-AC]

NSP-2,7-(OMHEG)₂-DMAE (16 mg, 14.8 umoles) in DMF (1.5 mL) was treatedwith diisopropylethylamine (5.2 uL, 2 equivalents) and TSTU (9 mg, 2equivalents). The reaction was stirred at room temperature for 30minutes and then cooled to 0° C. This solution was then added drop wiseto a solution of 6-aminocaproic acid (20 mg, 55.5 10 equivalents)dissolved in 0.1 M NaHCO₃ (2 mL). The resulting reaction was stirred at0° C. in an ice-bath and after 15 minutes it was warmed to roomtemperature. After 1 hour, the reaction was analyzed by HPLC using a C₁₈column from Phenomenex, 4.6 mm×30 cm, and a 30 minute gradient of10%→70% MeCN in water, each with 0.05% trifluoroacetic acid, at a flowrate of 1 mL/min, showed complete conversion to product eluting at 17.2minutes. The product was purified by preparative HPLC using a C₁₈, 20mm×30 cm column. The HPLC fraction, containing product was frozen at−80° C. and lyophilized to dryness. Yield=15.2 mg (84%).

(i) Synthesis of2′,6′-dimethyl-4′-N-succinimidyloxycaproylamidocarbonylphenyl-2,7-bis[O-methoxy(hexa)ethyleneglycol]-10-N-sulfopropyl-acridinium-9-carboxylate[NSP-2,7-(OMHEG)₂-DMAE-AC-NHS]

NSP-2,7-(OMHEG)₂-DMAE-AC (15 mg, 12,7 umoles) from step (h) wasdissolved in DMF (1.5 mL) and treated with diisopropylethylamine (3.3uL, 1.5 equivalents) and TSTU (4.6 mg, 1.2 equivalents). The reactionwas stirred at room temperature for 30 minutes and then analyzed by HPLCusing a C₁₈ column from Phenomenex, 4.6 mm×30 cm, and a 40 minutegradient of 10%→60% MeCN in water, each with 0.05% trifluoroacetic acid,at a flow rate of 1 mL/min, which, showed complete conversion to producteluting at 24.5 minutes. The product was purified by preparative HPLCusing a C₁₈, 20 mm×30 cm column. The HPLC fraction, containing productwas frozen at −80° C. and lyophilized to dryness. Yield=8 mg (50%).MALDI-TOF MS 1294.5 obs. (1293.5 calc.).

The following reactions describe the synthesis of 2,7-(OMHEG)₂-DMAE andNSP-2,7-(OMHEG)₂-DMAE, their NHS esters

The following reactions describe the synthesis ofNSP-2,7-(OMG)₂-DMAE-AC-NHS and NSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate-NHSester.

Example 10 Synthesis of 2′,6′-dimethyl-4′-carboxyphenyl2,7-bis[O-methoxytri(ethylene)glycol]-10-N-sulfopropyl-acridinium-9-carboxylate[NSP-2,7-(OMTEG)₂-DMAE], its NHS Ester and2′,6′-dimethyl-4′-N-succinimidyl-oxyglutarylamidohexa(ethylene)-glycolamido-carbonylphenyl-2,7-bis[O-methoxytri(ethylene)glycol]-10-sulfopropyl-acridinium-9-carboxylate[NSP-2,7-(OMTEG)₂-DMAE-HEG-glutarate-NHS]

These compounds were synthesized using the procedures described inExample 9 for the synthesis of NSP-2,7-(OMTEG)₂-DMAE-NHS ester usingmethoxytri(ethylene)glycol instead of methoxyhexa(ethylene)glycol.

The following reactions describe the synthesis ofNSP-2,7-(OMTEG)₂-DMAE-NHS ester andNSP-2,7-(OMTEG)₂-DMAE-HEG-glutarate-NHS ester.

Example 11 Synthesis of2′,6′-dimethyl-4′-N-succinimidyloxycapropyl-amidocarbonylphenyl-N¹⁰-2,7-tris[O-methoxyhexa(ethylene)glycol-sulfonamidylpropyl]-acridinium-9-carboxylate[N¹⁰-2,7-(OMHEG-SP)₃-DMAE-AC-NHS] (a) Synthesis of2′,6′-dimethyl-4′-carboxyphenyl-N¹⁰-2,7-tris[O-methoxyhexa(ethylene)glycol-sulfonamidylpropyl]-acridinium-9-carboxylate

NSP-2,7-(OSP)₂-DMAE (10 mg, 0.0129 mmol) was dissolved in methanol (5mL), and cooled in an ice-bath. Thionyl chloride (0.5 mL) was added dropwise and the reaction was stirred briefly in the ice-bath and thenwarmed to room temperature and stirred for 16 hours. HPLC analysis usinga C₁₈ column from Phenomenex, 4.6 mm×30 cm, and a 30 minute gradient of10%→70% MeCN in water, each with 0.05% trifluoroacetic acid, at a flowrate of 1 mL/min, showed complete conversion to product eluting at 12minutes. The solvent was removed under reduced pressure and the residuewas suspended in ethyl acetate (˜5 mL) and evaporated to dryness.

The methyl ester was then suspended in thionyl chloride (1.5 mL) andheated at reflux for 1 hour under a nitrogen atmosphere. It was thencooled to room temperature and the thionyl chloride was removed undervacuum. The residue was rinsed with anhydrous ether (˜5 mL) severaltimes and then dried under vacuum. It was then treated with a solutionof methoxy hexa(ethylene)glycol amine (synthesized using proceduresdescribed in U.S. Pat. No. 6,664,043 and Yamashita et al. J. Am. Chem.Soc. 1995, 117, 6249-6253) (0.1 mL) dissolved in dichloromethane (2 mL).

The reaction was stirred at room temperature. After one hour, HPLCanalysis indicated complete conversion to product eluting at 21 minutes.The solvent was then removed under reduced pressure and the crudeproduct was suspended in 1 N HCl (˜5 mL) and refluxed under a nitrogenatmosphere for 2 hours to hydrolyze the methyl ester. It was then cooledto room temperature and analyzed by HPLC which, indicated completehydrolysis of the methyl ester. The product was observed to elute at19.5 minutes.

The product was purified by preparative HPLC using a C₁₈ YMC, 20×300 mmcolumn using the above gradient and a solvent flow rate of 16 mL/min.The HPLC fraction containing product was frozen at −80° C. andlyophilized to dryness. Yield=3.8 mg (˜20%), MALDI-TOF MS 1602.92(calc.).

b) Synthesis of2′,6′-dimethyl-4′-carboxycaproylamidocarbonylphenyl-N¹⁰-2,7-tris[O-methoxyhexa(ethylene)glycol-sulfonamidylpropyl]-acridinium-9-carboxylate

A solution of N¹⁰-2,7-(OMHEG-SP)₃-DMAE (3.8 mg, 2.4 umoles) from inanhydrous DMF (1 mL) was treated with diisopropylethylamine (2.0 uL,2.4×5 umoles) and TSTU (4 mg, 2.4×5 umoles). The reaction was stirred atroom temperature for 30 minutes and then HPLC analysis using a C₁₈column from Phenomenex, 4.6 mm×30 cm, and a 30 minute gradient of10%→100% MeCN in water, each with 0.05% trifluoroacetic acid, at a flowrate of 1 mL/min, showed complete conversion to product eluting at 16.2minutes (starting material elutes at 15.3 minutes). The DMF solution wascooled in an ice-bath and then added in 0.1 mL portions to an ice-coldsolution of 6-aminocaproic acid (6 mg, 47.4 umoles) dissolved in 0.1 Msodium bicarbonate (1 mL). The reaction was then warmed to roomtemperature and stirred for an hour. HPLC analysis indicated completeconversion to product eluting at 15.4 minutes. The product was purifiedby preparative HPLC using a C₁₈ YMC 20×300 mm column. The HPLC fractionwas frozen at −80° C. and lyophilized to dryness. Yield=3.3 mg (81%);MALDI-TOF MS 1719 obs. (1717.1 calc.).

c) Synthesis of2′,6′-dimethyl-4′-N-succinimidyloxycapropylamidocarbonylphenyl-N¹⁰-2,7-tris[O-methoxyhexa(ethylene)glycolsulfonamidylpropyl]-acridinium-9-carboxylate

A solution of N¹⁰-2,7-(OMHEG-SP)₃-DMAE-AC (3.3 mg, 2 umoles) from step(a) in anhydrous DMF (1 mL) was treated with diisopropylethylamine (1.8uL, 10 umoles) and TSTU (3 mg, 10 umoles). The reaction was stirred atroom temperature after 15 minutes, HPLC analysis as described above insection (a) showed complete conversion to product eluting at 16.2minutes. The product was purified by preparative HPLC and the HPLCfraction was frozen at −80° C. and lyophilized to dryness. Yield=2.2 mg(63%); MALDI-TOF MS (1814.1 calc.)

The following reactions describe the synthesis ofN¹⁰-2,7-(OMHEG-SP)₃-DMAE-AC-NHS.

Example 12 Synthesis of2′,6′-dimethyl-4′-N-succinimidyloxyglutaryl-amidohexa(ethylene)glycol-amidocarbonylphenyl-N¹⁰-methoxyhexa(ethylene)glycol-sulfonamidylpropyl-2,7-dimethoxy-acridinium-9-carboxylate[N¹⁰-(OMHEG-SP)-2,7-(OMe)₂-DMAE-HEG-Glutarate-NHS] (a) Synthesis of2′,6′-dimethyl-4′-carboxyphenyl-N¹⁰-methoxyhexa(ethylene)glycol-sulfonamidylpropyl]-2,7-dimethoxy-acridinium-9-carboxylate

NSP-2,7-dimethoxy-DMAE benzyl ester (22 mg) from Example 1 was dissolvedin thionyl chloride and the solution was heated in an oil-bath at 55° C.under a nitrogen atmosphere for 4 hours. The thionyl chloride wasremoved under reduced pressure and the residue was rinsed with anhydrousether and dried under vacuum. This residue was treated with a solutionof methoxy hexa(ethylene)glycol amine (0.1 mL) in dichloromethane (2mL). The reaction was stirred at room temperature. After 30 minutes,HPLC analysis-using a C₁₈ column from Phenomenex, 4.6 mm×30 cm, and a 30minute gradient of 10%→100% MeCN in water, each with 0.05%trifluoroacetic acid, at a flow rate of 1 mL/min, showed completeconversion to product eluting at 19 minutes. The solvent was themremoved under reduced pressure and the resulting oil was stirred in 30%HBr/AcOH (2 mL) at room temperature for 6 hours to effect thedebenzylation of the carboxylic acid. Ether (10 mL) was then added and adark red oil separated out. The ether was decanted and the oil wasrinsed several times with ether. It was then dissolved in DMF (2-3 mL)and analyzed by HPLC which, indicated product eluting at 15 minutes. Theproduct was purified by preparative HPLC using a YMC C₁₈, 20×300 mmcolumn. The HPLC fractions containing product were frozen at −80° C. andlyophilized to dryness. Yield=5.3 mg (19%); MALDI-TOS MS 831.9 calc.

(b) Synthesis of2′,6′-dimethyl-4′-aminohexa(ethylene)glycolamidocarbonylphenyl-N¹⁰-methoxyhexa(ethylene)glycol-sulfonamidylpropyl2,7-dimethoxy-acridinium-9-carboxylate

A solution of N¹⁰-(OMHEG-SP)-2,7-(OMe)₂-DMAE (5.3 mg, 6.4 umoles) fromstep (a) in anhydrous DMF (1 mL) was treated with diisopropylethylamine(5.6 uL, 17 umoles) and TSTU (6 mg, 10.2 umoles). The reaction wasstirred at room temperature. After 30 minutes, HPLC analysis asdescribed above in section (a) indicated complete conversion to producteluting at 16.3 minutes. The reaction mixture was added drop wise to asolution of diamino hexa(ethylene)glycol (10 mg, 0.0354 mmol) in DMF(0.5 mL). The reaction was stirred at room temperature for 30 minutesand then analyzed by HPLC which, indicated complete conversion toproduct eluting at 14 minutes. The product was purified by preparativeHPLC using a YMC C₁₈, 20×300 mm column. The HPLC fraction was frozen at−80° C. and lyophilized to dryness. Yield=3.7 mg (53%).

(c) Synthesis of2′,6′-dimethyl-4′-N-succinimidyloxyglutarylamidohexa(ethylene)-glycolamidocarbonylphenyl-N¹⁰-methoxyhexa(ethylene)glycol-sulfonamidylpropyl2,7-dimethoxy-acridinium-9-carboxylate

A solution of N¹⁰-(OMHEG-SP)-2,7-(OMe)₂-DMAE-HEG-NH₂ (3.7 mg, 3.4umoles) from step (b) in anhydrous methanol (1 mL) was treated withdiisopropylethylamine (3 uL, 17 umoles) and glutaric anhydride (2 mg, 17umoles). The reaction was stirred at room temperature and after 30minutes, HPLC analysis as described in section (a) indicated completeconversion to product eluting at 14.6 minutes. The reaction mixture wasevaporated to dryness. The residue was dissolved in DMF (1 mL) andtreated with diisopropylethylamine (3 uL) and TSTU (6 mg, 17 umoles).The reaction was stirred at room temperature and after 30 minutes, HPLCanalysis indicated product eluting at 15.3 minutes. The product waspurified by preparative HPLC using a C₁₈ YMC, 20×300 mm column. The HPLCfraction containing product was frozen at −80° C. and lyophilized todryness. Yield=1.7 mg (39%). MALDI-TOF-MS 1303.5 calc.

The following reactions describe the synthesis ofN¹⁰-(OMHEG-SP)-2,7-(OMe)₂-DMAE-HEG-Glutarate-NHS.

Example 13 Synthesis of3-[9-({5-carboxypentyl)[4-methylphenyl)sulfonyl]amino}carbonyl)-10-acridiniumyl]-1-propanesulfonate(NSP-AS) and its N-succinimidyl ester (NSP-AS-NHS)

NSP-AS was synthesized using the procedures reported by Adamczyk et al.,Tetrahedron, vol. 55, pp. 10899-10914 (1999) as follows. NSP-AS (8.8 mg,14.4 umoles) was dissolved in anhydrous DMF (I mL) and was treated withdiisopropylethylamine (5 uL, 2 equivalents) and TSTU (8.2 mg, 1.5equivalents). The reaction was stirred at room temperature. After 30minutes HPLC analysis using a C₁₈ column from Phenomenex, 4.6 mm×30 cm,and a 30 minute gradient of 10%→70% MeCN in water, each with 0.05%trifluoroacetic acid, at a flow rate of 1 mL/min, showed completeconversion to product eluting at 17.7 minutes with no starting materialat 15.5 minutes. The product was purified by preparative HPLC using aC₁₈, 20 mm×30 cm column. The product fraction, containing product wasfrozen at −80° C. and lyophilized to dryness. Yield=5-8 mg (57%)

The following reactions describe the synthesis of NSP-AS-NHS ester.

Example 14 Synthesis of3-[9-({5-carboxypentyl)[4-methylphenyl)sulfonyl]amino}carbonyl)-2,7-dimethoxy-10-acridiniumyl]-1-propanesulfonate(NSP-2,7-dimethoxy-AS) and its N-succinimidyl ester (NSP-AS-NHS)

These compounds were synthesized using the procedures and referencecited in Examples 7-13.

The following reactions describe the synthesis of NSP-2,7-(OMe)₂-AS andits NHS ester.

Example 15 Synthesis of NSP-2,7-(OMHEG)₂-DMAE-HEG-Theophylline Conjugate

A solution of 8-carboxypropyltheophylline (Sigma, 5 mg, 18.7 umoles) inanhydrous DMF (1 mL) was treated with diisopropylethylamine (3.2 uL, 1equivalent) and HATU (7 mg, 1 equivalent). The reaction was stirred atroom temperature for 10 minutes and then a solution ofNSP-2,7-(OMHEG)₂-DMAE-HEG-NH₂ (5 mg, 3.7 umoles) from Example 9, step(f) was added in anhydrous DMF (1 mL) along with diisopropylethylamine(2 uL). This reaction was stirred at room temperature. After 16 hoursHPLC analysis-using a C₁₈ column from Phenomenex, 4.6 mm×30 cm, and a 30minute gradient of 10%→70% MeCN in water, each with 0.05%trifluoroacetic acid, at a flow rate of 1 mL/min, showed completeconversion to product eluting at 18 minutes. The product was purified bypreparative HPLC using a C₁₈ YMC, 20×300 mm column. The HPLC fractioncontaining product was frozen at −80° C. and lyophilized to dryness.Yield=4.0 mg (67%). MALDI-TOF-MS 1593.8 calc. 1596.7 obs.

The following reactions describe the synthesis ofNSP-2,7-(OMHEG)₂-DMAE-HEG-Theophylline.

Example 16 General Procedure for Protein Labeling with AcridiniumCompounds

The anti-TSH murine monoclonal antibody (1 mg, 6.67 nmoles, stocksolution 5 mg/mL, 0.2 mL) was diluted with 300 uL of 0.1 M sodiumphosphate, pH 8. The protein solutions were treated with DMF solutionsof various acridinium esters as follows:

-   a) For labeling with 20 equivalents NSP-DMAE-HEG-Glutarate-NHS    ester, 63 uL of a 2 mg/mLDMF solution of the compound was added;-   b) for labeling with 20 equivalents of NSP-2,7-(OMTEG)₂-DMAE-NHS    ester, 46 uL of a 2.67 mg/mL DMF solution of the compound was added;-   c) for labeling with 20 equivalents of    NSP-2,7-(OMTEG)₂-DMAE-HEG-Glutarate-NHS ester, 52 uL of a 3.33 mg/mL    DMF solution was added;-   d) for labeling with 20 equivalents of NSP-2,7-(OMHEG)₂-DMAE-AC-NHS    ester, 43 uL of a 4 mg/mL DMF solution was added and;-   e) for labeling with 20 equivalents of    NSP-2,7-(OMHEG)2-DMAE-HEG-Glutarate-NHS ester, 52 uL of a 3.33 mg/mL    DMF solution was added.

All reactions were stirred at 4° C. for 16 hours and were thentransferred to 2 mL amicon filters(MW 30,000 cutoff) and diluted with1.5 mL de-ionized water. The volume was reduced to ˜0.1 mL bycentrifuging at 4500 G. The concentrated conjugate solutions werediluted with 2 mL de-ionized water and centrifuged again to reduce thevolume. This process was repeated a total of four times. Finally, theconcentrated conjugates were diluted with 0.1 mL de-ionized water.

These solutions were used for MALDI-TOF (Matrix-Assisted LaserDesorption Ionization-Time of Flight) mass spectral analysis, using theVoyager-DE instrument from Perkin-Elmer, to measure acridinium compoundincorporation. Typically, this entailed measuring the molecular weightof the unlabeled antibody and the labeled antibody. The acridiniumcompound label contributed the difference in mass of these twomeasurements. By knowing the molecular weight of the specific acridiniumcompound label, the extent of label incorporation of that specificacridinium compound could thus be calculated.

The number of labels per antibody molecule for NSP-DMAE-HEG-glutarate,NSP-2,7-(OMTEG)₂-DMAE, NSP-2,7-(OMTEG)₂-DMAE-HEG-glutarate,NSP-2,7-(OMHEG)₂-DMAE-AC and NSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate were 8,8, 7, 10 and 8 respectively.

Protein concentrations were determined by a commercial calorimetricassay.

Chemiluminescence from the conjugates was measured by first diluting theconjugates in 10 mM phosphate pH 8 containing 150 mM NaCl, 0.05% BSA and0.01% sodium azide and then conducting measurements in aACS:180®-Automated Chemiluminescent Immunoassay System instrument fromBayer Diagnostics. A typical measurement involves triggeringchemiluminescence from 25 uL of the diluted conjugate solution with theaddition of two reagents. Reagent 1 is a solution of 0.5% hydrogenperoxide in 0.1 N nitric acid. Reagent 2 is 0.25 N sodium hydroxide.Light was measured for a total of 5 seconds. Under theseconditions, >90% of the light from each sample was collected. The outputfrom luminometer instrument is expressed as RLUs (Relative Light Units).These values were normalized to that of NSP-DMAE which was assigned arelative chemiluminesence quantum yield of 1.0.

Labeling reactions with other acridinium compounds were carried out in asimilar manner.

Example 17 Chemiluminescence and Emission Wavelength Measurements ofAcridinium Compounds

Chemiluminescence from the various acridinium compounds was measured asdescribed for the protein conjugates in Example 16. A solution,typically 1 mg/mL in DMF, of the various HPLC-purified compounds, wasserially diluted into 10 mM phosphate pH 8 containing 150 mM NaCl, 0.05%BSA and 0.01% sodium azide and then chemiluminescence measurements wereconducted in a luminometer (MLA1™, Bayer Diagnostics). The relativequantum yield of NSP-DMAE was assigned a value of 1.0.

Emission wavelength from the acridinium compounds was measured usingFSSS (Fast Spectral Scanning System) camera from Photoresearch Inc. In atypical measurement, 25-50 uL of a 1 mg/mL DMF solution of theacridinium compound was diluted with DMF (˜0.3 mL). Chemiluminescencewas triggered by the addition of the two reagents described in example10. Just prior to the addition of the second reagent, the shutter of thecamera was opened and light was collected for 5 seconds. The output ofthe instrument is a graph of light intensity versus wavelength. Emissionmaxima for each compound are listed in Table 1.

Example 18 Theophylline Immunoassay Using a High Quantum YieldAcridinium Compound as Chemiluminescent Label

A high quantum yield acridinium compound was tested as a label in acompetitive immunoassay for comparison with a control acridiniumcompound used separately as a label in the same competitive immunoassay.A label is an atom, molecule, compound, ion, radical or macromolecule,that when conjugated or complexed to a ligand, or when conjugated orcomplexed to a receptor, enables qualitative or quantitativeidentification, assessment, characterization, detection or measurementof an analyte or analytes in an assay.

In this example the high quantum yield acridinium compoundNSP-2,7-(OMHEG)₂-DMAE-HEG was the label to be tested and to be comparedto the control acridinium compound NSP-DMAE-HEG, which is the actuallabel in the commercially marketed Bayer Diagnostics ACS:180®Theophylline Assay. The control acridinium compound NSP-DMAE-HEG isreferred to as a control because it is the actual label in thecommercially available product, the Bayer Diagnostics ACS:180®Theophylline Assay.

The high quantum yield acridinium compound NSP-2,7-(OMHEG)₂-DMAE-HEG hasa higher quantum yield than the control acridinium compoundNSP-DMAE-HEG, which has a lower quantum yield. When used as a label inthe Bayer Diagnostics ACS:180® Theophylline Assay, the high quantumyield acridinium compound NSP-2,7-(OMHEG)₂-DMAE-HEG was expected toenhance the Bayer Diagnostics ACS:180® Theophylline Assay when comparedwith the lower quantum yield control acridinium compound NSP-DMAE-HEG,when also used separately as a label in the Bayer Diagnostics ACS:180®Theophylline Assay.

An assay, which is synonymous with test, is a method, a reaction or theact of qualitative or quantitative identification, assessment,characterization, detection or measurement of the properties or amountof a substance, substances, analyte or analytes or their parts.

The Bayer Diagnostics ACS:180® Theophylline Assay is one of a series ofcommercially marketed immunoassays manufactured by Bayer Diagnostics forapplication on the Bayer Diagnostics ACS:180® (AutomatedChemiluminescent Immunoassay System).

An immunoassay, which is synonymous with immunologic assay orimmunochemical assay, is a method, a reaction or the act of qualitativeor quantitative identification, assessment, characterization, detectionor measurement of the properties or amount of a substance, substances,analyte or analytes or their parts by action of an antigen, epitope,hapten, ligand, carrier, or macromolecule with a receptor that isusually an antibody or other biological receptor.

The Bayer Diagnostics ACS:180® Theophylline Assay is a competitiveimmunoassay which uses a chemiluminescent acridinium compound conjugateof theophylline, which is NSP-DMAE-HEG-theophylline, for measurement oftheophylline in a sample.

A sample is a material, which may contain an analyte or analytes. Inreference to the Bayer Diagnostics ACS:180® Theophylline Assay, thesample was serum, which might contain analyte.

An analyte is a substance or substances, which may be present in asample, and which can be qualitatively or quantitatively identified,assessed, characterized, detected or measured in an assay. In referenceto the Bayer Diagnostics ACS:180® Theophylline Assay the analyte istheophylline.

Both the high quantum yield acridinium compoundNSP-2,7-(OMHEG)₂-DMAE-HEG and the control acridinium compoundNSP-DMAE-HEG were labels conjugated to a ligand. A ligand is an atom,molecule, compound, ion or radical that binds specifically to amacromolecule: that macromolecule being a receptor. In reference to theBayer Diagnostics ACS:180® Theophylline Assay the ligand istheophylline.

An acridinium compound conjugated to a ligand is a tracer. A tracer is aconjugate or complex of a label with a ligand, or is a conjugate orcomplex of a label with a receptor, which by action of the label,enables qualitative or quantitative identification, assessment,characterization, detection or measurement of an analyte or analytes inan assay.

In the Bayer Diagnostics ACS:180® Theophylline Assay reagents were mixedwith the sample to start the assay reaction. A reagent is a substance orsubstances which have chemical action with another substance or othersubstances, but more specifically with respect to an assay, an assayreagent is a substance or substances, other than and not including thesample, which will react with an analyte, if the analyte is present in asample.

In reference to the Bayer Diagnostics ACS:180® Theophylline Assay therewere two assay reagents. The first assay reagent was the solid phase.The solid phase is an assay reagent consisting of a ligand or a receptorconjugated to or complexed to, directly or indirectly to a separablematerial. In reference to the Bayer Diagnostics ACS:180® TheophyllineAssay the solid phase is a receptor on magnetically separableparamagnetic particles.

A receptor is a macromolecule, which specifically binds one or moreanalytes, antigens, atoms, compounds, epitopes, haptens, ions, ligands,molecules, radicals, tracers or other receptors. In reference to theBayer Diagnostics ACS:180® Theophylline Assay, the receptor isanti-theophylline antibody which binds both the analyte, which istheophylline, and the tracer.

The second assay reagent is the tracer. In reference to the BayerDiagnostics ACS:180® Theophylline Assay, the tracer, which is synonymouswith Lite Reagent, is a conjugate of a label, which is an acridiniumcompound, and a ligand, which like the analyte is theophylline. Since,in reference to the Bayer Diagnostics ACS:180® Theophylline Assay, theligand is theophylline and is the same as the analyte, which is alsotheophylline, both the analyte and the tracer will bind to the receptorin the reaction mixture.

The reaction mixture is a combining of reagents, but more specificallywith respect to an assay, the assay reaction mixture is a combining ofsample and assay reagents in an assay or assay reaction. A reaction isthe action of a substance or substances on another substance or othersubstances, but more specifically with respect to an assay, the assayreaction is the action of assay reagents on a sample in an assay.

In reference to the Bayer Diagnostics ACS:180® Theophylline Assay, theassay reaction mixture contains sample, solid phase and tracer, and theassay reaction is the action of the solid phase and tracer on thesample, which may contain analyte.

In reference to the Bayer Diagnostics ACS:180® Theophylline Assay, theassay reaction is the binding of both the analyte, which is theophyllineif present in the sample, and the tracer, which is the acridiniumcompound conjugate of the ligand theophylline, to the same solid phase,which binds either an analyte or a tracer at any one receptor, but doesnot bind both an analyte and a tracer at the same time at the samereceptor.

Since the solid phase has a smaller amount of receptor, with respect tothe total amount of analyte, when present in the sample, then the totalof the amount of analyte plus tracer in the assay reaction mixture isgreater than the amount of the receptor on the solid phase. Thus thetotal of the amount of analyte plus tracer cannot bind to the solidphase in totality.

Consequently, the presence of analyte in a sample will block or competewith the binding of the tracer to the solid phase in the assay reactionmixture, where the amount of blocking or competition depends on theamount of analyte present in the sample.

Therefore, the amount of analyte in a sample is inversely correlated tothe amount of tracer that will bind to the solid phase in an assayreaction mixture. In reference to the Bayer Diagnostics ACS:180®Theophylline Assay, the analyte, which is theophylline if present in thesample, blocks or competes with the tracer, which is an acridiniumcompound conjugate of theophylline, for a limited amount ofanti-theophylline antibody, which is the receptor on magneticallyseparable paramagnetic particles.

The Bayer Diagnostics ACS:180® automatically performed the followingsteps for the Bayer Diagnostics ACS:180® Theophylline Assay. First,0.020 mL of each of fourteen samples was dispensed into a separatecuvet. A cuvet is an optically transparent or translucent container thatholds the assay reaction mixture and in which the assay reaction takesplace.

The fourteen samples each contained separate known amounts oftheophylline. The amounts of theophylline given as concentrations ineach of these fourteen samples were 0, 1.40, 2.10, 2.80, 4.20, 5.60,9.21, 15.6, 32,7, 68.3, 129, 288, 500, and 1000 micromolar. The amountsof theophylline given as numbers of molecules in each of these samefourteen samples were 0, 0.028, 0.042, 0.056, 0.084, 0.112, 0.184,0.313, 0.655, 1.37, 2,59, 5.76, 10.0, and 2.00 picomoles, respectively.

Next, the Bayer Diagnostics ACS:180® dispensed two assay reagentstogether into each cuvet and mixed the assay reagents with the samplewithin each cuvet. The first of the two assay reagents was 0.450 mL ofsolid phase, which contained 8.7 picomoles of anti-theophylline antibodyon magnetically separable paramagnetic particles.

The second of the two assay reagents was 0.100 mL of tracer, which was0.026 picomole of acridinium compound conjugated to theophylline. Boththe high quantum yield acridinium compound NSP-2,7-(OMHEG)₂-DMAE-HEG andthe control acridinium compound NSP-DMAE-HEG were tested separately aslabels conjugated to theophylline, in the form of two tracers:NSP-2,7-(OMHEG)₂-DMAE-HEG-theophylline and NSP-DMAE-HEG-theophylline,respectively.

The assay reaction proceeded for 7.5 minutes at 37° C. The BayerDiagnostics ACS:180® finished the ACS:180® Theophylline Assay bymagnetically separating the solid phase from other assay reagents, thenremoving fluid from the cuvet and then washing the solid phase in thecuvet with water.

Chemiluminescence from acridinium compound on the solid phase wasinitiated with subsequent light emission with sequential additions of0.30 mL each of Bayer Diagnostics ACS:180® Reagent 1 and BayerDiagnostics ACS:180® Reagent 2. Bayer Diagnostics ACS:180® Reagent 1 was0.1 M nitric acid and 0.5% hydrogen peroxide. Bayer Diagnostics ACS:180®Reagent 2 was 0.25 M sodium hydroxide and 0.05% cetyltrimethylanmuoniumchloride.

The Bayer Diagnostics ACS:180® measured the chemiluminescence in eachcuvet with each cuvet corresponding to a single assayed sample. TheBayer Diagnostics ACS:180® measured the chemiluminescence as relativelight units (RLUs). In reference to the Bayer Diagnostics ACS:180®Theophylline Assay, the amount of analyte is inversely correlated to theamount of tracer that will bind to the solid phase.

Consequently, the amount of analyte is inversely correlated to thenumber of RLUs measured by the Bayer Diagnostics ACS:180®. This meansthat the greater the amount of the analyte theophylline in a sample thenfewer RLUs are measured with respect to lower amounts of the analytetheophylline in a sample where more RLUs are measured.

Normalization to percentage of chemiluminescence measured in the absenceof analyte was calculated for comparison of the relativechemiluminescence given for each amount of analyte for the high quantumyield acridinium compound, NSP-2,7-(OMHEG)₂-DMAE-HEG, compared with thecontrol acridinium compound NSP-DMAE-BEG.

The greater the spacing between chemiluminescence values for successiveamounts of analyte is an indicator of enhanced sensitivity. The greaterthe chemi-luminescence difference between small amounts of analyte fromthe chemiluminescence obtained in the absence of analyte, permits betterdifferentiation of small amounts of analyte and the absence of analytein competitive assay, thereby enhancing sensitivity.

In reference to the Bayer Diagnostics ACS:180® Theophylline Assayrelative to the lower quantum yield label NSP-DMAE-HEG, the high quantumyield acridinium compound NSP-2,7-(OMHEG)₂-DMAE-HEG generated greaterdifferentiation between small amounts of theophylline and the absence oftheophylline when used as a competitive immunoassay label.

The slope of the line generated for each tracer using the BayerDiagnostics ACS:180® Theophylline Assay is an indicator of sensitivity.The greater the absolute magnitude of the slope of the line for aparticular tracer or label in a competitive immunoassay, the moredistant is the chemiluminescence for a particular amount of analyte fromthe chemiluminescence from a sample with no analyte and the assay isbetter able to measure the difference between the presence of analyteand the absence of analyte.

In reference to the Bayer Diagnostics ACS:180® Theophylline Assay, thehigh quantum yield acridinium compound label NSP-2,7-(OMHEG)₂-DMAE-HEGgave enhanced slope relatives to the control acridinium compound labelNSP-DMAE-HEG.

The greater absolute magnitude of the slope generated by the highquantum yield acridinium compound label for both the high amounts andparticularly the low amounts of theophylline, relative to the controlacridinium compound and NSP-DMAE-HEG, indicates an enhancement ofsensitivity for the Bayer Diagnostics ACS:180® Theophylline Assay usinghigh quantum yield acridinium compounds as immunoassay labels.

For the Bayer Diagnostics ACS:180® Theophylline Assay, which is acompetitive immunoassay, sensitivity is the least measurable non-zeroamount of analyte. The least measurable non-zero amount of analyte inthe Bayer Diagnostics ACS:180® Theophylline Assay is the amount ofanalyte corresponding to the greatest measured chemiluminescence that isless than the difference of the chemiluminescence measured in theabsence of analyte minus two standard deviations of chemiluminescencemeasured in the absence of analyte.

For example, in competitive immunoassays where the followingrepresentations are given:

n=positive integer greater than 0.

x=the measured amount of analyte corresponding to y, where x0<x1<x2<x3<. . . <xn are successively greater measured amounts of analyte.

y=the chemiluminescence measured for an amount of analyte, representedby x, where y0>y1>y2>y3> . . . >yn are successively lesser values ofchemiluminescence, measured for x0<x1<x2<x3< . . . <xn, respectively.

x0=a zero amount of analyte or the amount of analyte equal to zero.

y0=the chemiluminescence measured for an amount of analyte equal tozero, which is x0.

s=one standard deviation of y0.

Then the sensitivity=xn for yn<y0−2s, when n=the least, positive,nonzero integer.

The sensitivity for the Bayer Diagnostics ACS:180® Theophylline Assayusing the high quantum yield acridinium compoundNSP-2,7-(OMHEG)₂-DMAE-HEG was 1.4 μM. The sensitivity for the BayerDiagnostics ACS:180® Theophylline Assay using the control acridiniumcompound NSP-DMAE-HEG was 4.2 μM. The quotient of 4.2 μM and 1.4 μM is3.

The Bayer Diagnostics ACS:180® Theophylline Assay using the high quantumyield acridinium compound as a label measured an amount of theophyllinethat was three-fold smaller than the commercially marketed BayerDiagnostics ACS:180® Theophylline Assay that used the lower quantumyield control acridinium compound as a label.

The high quantum yield acridinium compound NSP-2,7-(OMHEG)₂-DMAE-HEGenhanced the sensitivity of the Bayer Diagnostics ACS:180® TheophyllineAssay three-fold when compared to the control acridinium compoundNSP-DMAE-HEG.

The example establishes that when used as chemiluminescent immunoassaylabels the enhanced chemiluminescent light emission from high quantumyield acridinium compounds enhances the sensitivity of competitiveimmunoassays.

Example 19 TSH Immunoassay Using High Quantum Yield Acridinium Compoundsas Chemiluminescent Labels

High quantum yield acridinium compounds were tested as labels in asandwich immunoassay for comparison with control acridinium compoundsused separately as labels in the same sandwich immunoassay.

In this example the high quantum yield acridinium compounds,NSP-2,7-(OMTEG)₂-DMAE, NSP-2,7-(OMTEG)₂-DMAE-HEG-glutarate,NSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate and NSP-2,7-(OMHEG)₂-DMAE-AC werethe chemiluminescent labels to be tested and to be compared to thecontrol acridinium compound NSP-DMAE-HEG-glutarate.

The high quantum yield acridinium compounds have higher quantum yieldsthan the control acridinium compound NSP-DMAE-HEG-glutarate, and wereexpected to enhance the Bayer Diagnostics ACS:180® TSH3 Assay.

The Bayer Diagnostics ACS:180® TSH3 Assay is a sandwich immunoassaywhich uses a chemiluminescent acridinium compound conjugate of anti-TSHantibody, for measurement of TSH (Thyroid Stimulating Hormone) in asample.

A sample is a material, which may contain an analyte or analytes. Inreference to the Bayer Diagnostics ACS:180® TSH3 Assay, the sample wasserum, which might contain analyte. An analyte is a substance orsubstances, which may be present in a sample, and which can bequalitatively or quantitatively identified, assessed, characterized,detected or measured in an assay.

In reference to the Bayer Diagnostics ACS:180® TSH3 Assay, the analyteis TSH. Both the high quantum yield acridinium compounds and the controlacridinium compound NSP-DMAE-HEG-glutarate were labels conjugated to aantibodies.

In reference to the Bayer Diagnostics ACS:180® TSH3 Assay, there are twoantibodies, one of which is labeled with the acridinium compound and iscalled the tracer while the other is covalently attached to paramagneticparticles (PMP) solid phase.

In the ACS:80® TSH3 Assay, the assay mixture contains sample, tracer andsolid phase, and the assay reaction is the action of the tracer andsolid phase on the sample, which may contain analyte. Since the tracerbinds to the analyte and the analyte is bound to the solid phase, athree-part ‘sandwich’ is formed of tracer, analyte and solid phase.

Consequently, the presence of analyte in a sample will cause the bindingof the tracer through the analyte to the solid phase in the assayreaction mixture, where the amount of tracer bound to the solid phasedepends on the amount of analyte present in the sample.

The Bayer Diagnostics ACS:180® automatically performed the followingsteps for the TSH3 Assay. First, 200 μL of each of twelve samples wasdispensed into a separate cuvet. A cuvet is an optically transparent ortranslucent container that holds the assay reaction mixture and in whichthe assay reaction takes place. The twelve samples each containedseparate known amounts of TSH.

The amounts of TSH given as concentrations in each of these twelvesamples were 0, 0.002, 0.004, 0.010, 0.015, 0.020, 0.025, 0.030, 0.10,1.0, 10 and 100 mIU/L. Next, the Bayer Diagnostics ACS:180® dispensedtwo assay reagents together into each cuvet and mixed the assay reagentswith the sample within each cuvet.

The first of the two assay reagents was 0.100 mL of tracer, whichcontained 0.22 picomoles of anti-TSH antibody conjugated with acridiniumcompound. Both the high quantum yield acridinium compounds and thecontrol acridinium compounds NSP-DMAE-HEG-glutarate were testedseparately as labels conjugated to anti-TSH antibody. The assay reactionproceeded for 2,5 minutes at 37° C.

The second of the two assay reagents was 0.225 mL of solid phase, whichwas anti-TSH antibody conjugated to paramagnetic microparticles. Theassay reaction proceeded for 5.0 minutes at 37° C. The assay reactionproceeded for a total of 7.5 minutes at 37° C.

The Bayer Diagnostics ACS:180® finished the ACS:180® TSH3 Assay bymagnetically separating the solid phase from other assay reagents, thenremoving fluid from the cuvet and then washing the solid phase in thecuvet with water.

Chemiluminescence from acridinium compound on the solid phase wasinitiated with subsequent light emission with sequential additions of0.30 mL each of Bayer Diagnostics ACS:180® Reagent 1 and BayerDiagnostics ACS:180® Reagent 2. Bayer Diagnostics ACS:180® Reagent 1 was0.1 M nitric acid and 0.5% hydrogen peroxide. Bayer Diagnostics ACS:180®Reagent 2 was 0.25 M sodium hydroxide and 0.05% cetyltrimethylammoniumchloride.

The Bayer Diagnostics ACS:180® measured the chemiluminescence asrelative light units (RLUs) in each cuvet with each cuvet correspondingto a single assayed sample. In the assay, the amount of analyte iscorrelated to the amount of tracer that will bind to the solid phase.

Consequently, the amount of analyte is correlated to the number of RLUsmeasured by the Bayer Diagnostics ACS:180®. This means that the greaterthe amount of the analyte TSH in a sample, then the greater the amountof RLUs are measured with respect to lower amounts of the analyte TSH ina sample where fewer RLUs are measured.

The high quantum yield acridinium compound labels NSP-2,7-(OMTEG)₂-DMAE,NSP-2,7-(OMTEG)₂-DMAE-HEG-glutarate, NSP-2,7-(OMHEG)₂-DMAE-AC andNSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate gave enhanced chemiluminescence forall amounts of analyte.

Noise is the portion of chemiluminescence in a sandwich immunoassay of asample which is due to tracer that binds nonspecifically to the solidphase and which is measured in samples that contain no analyte. Signalis the portion of the chemiluminescence due to the specific binding ofthe tracer to the solid phase when analyte is present in the sample.

The total chemiluminescence measured in the Bayer Diagnostics ACS:180®TSH3 Assay for samples that do contain analyte is the sum of signal plusnoise, where signal is calculated as the difference of the totalchemiluminescence minus the noise.

Assay sensitivity is often defined as the least measurable amount ofanalyte. For the Bayer Diagnostics ACS:180® TSH3 Assay, which is asandwich immunoassay, sensitivity is the least measurable non-zeroamount of analyte. The least measurable non-zero amount of analyte isthe amount of analyte corresponding to the least measuredchemiluminescence that is greater than the sum of the noise plustwo-standard deviations of the noise.

In the Bayer Diagnostics ACS:180® TSH3 Assay signal and noise weredetermined for each tested tracer. The ratio of the signal divided bythe noise in a sandwich immunoassay for a particular amount of analyteis an indicator of sandwich immunoassay sensitivity. The greater thesignal to noise ratio for a particular amount of analyte in a sandwichimmunoassay, the more distant is the corresponding signal from the noiseand the better able is the assay to measure the difference between thesignal and the noise.

In the current assay, the high quantum yield acridinium compound labelsNSP-2,7-(OMTEG)₂-DMAE, NSP-2,7-(OMTEG)₂-DMAE-HEG-glutarate,NSP-2,7-(OMHEG)₂-DMAE-AC and NSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate gaveenhanced signal to noise ratios for amounts of analyte relative to thecontrol acridinium compound label NSP-DMAE-HEG-glutarate.

The greater signal to noise ratios generated by the high quantum yieldacridinium compound labels for both the high amounts and particularlythe low amounts of TSH, relative to the control acridinium compoundsDMAE and NSP-DMAE-HEG-glutarate, indicate an enhancement of sensitivityfor the Bayer Diagnostics ACS:180® TSH3 Assay using high quantum yieldacridinium compound as immunoassay labels.

The slope of the line generated for each tracer using the BayerDiagnostics ACS:180® TSH3 Assay is an indicator of sensitivity. Thegreater the slope of the line for a particular tracer or label in asandwich immunoassay, the more distant is the signal for a particularamount of analyte from the noise and the better able is the assay tomeasure the difference between the signal and the noise.

In the current assay, the high quantum yield acridinium compound labelsNSP-2,7-(OMTEG)₂-DMAE, NSP-2,7-(OMTEG)₂-DMAE-HEG-glutarate,NSP-2,7-(OMHEG)₂-DMAE-AC and NSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate gaveenhanced slopes relative to the control acridinium compound labelNSP-DMAE-HEG-glutarate.

The greater slopes generated by the high quantum yield acridiniumcompound labels for both the high amounts and particularly the lowamounts of TSH, relative to the control acridinium compounds DMAE andNSP-DMAE-HEG-glutarate, indicate an enhancement of sensitivity for theBayer Diagnostics ACS:180® TSH3 Assay using high quantum yieldacridinium compound as immunoassay labels.

The sensitivities measured for the Bayer Diagnostics ACS:180® TSH3 Assayusing the high quantum yield acridinium compound labelsNSP-2,7-(OMTEG)₂-DMAE, NSP-2,7-(OMTEG)₂-DMAE-HEG-glutarate,NSP-2,7-(OMHEG)₂-DMAE-AC and NSP-2,7-(OMHEG)₂-DMAE-HEG-glutarate wereenhanced over the sensitivities measured using for the controlacridinium compound NSP-DMAE-HEG-glutarate.

Assay sensitivity is often defined as the least measurable amount ofanalyte. In the current sandwich immunoassay the least measurablenon-zero amount of analyte is the amount of analyte corresponding to theleast measured chemiluminescence that is greater than the sum of thenoise plus two-standard deviations of the noise.

For example, in sandwich immunoassays where the followingrepresentations are given:

n=positive integer greater than 0.

x=the measured amount of analyte corresponding to y, where x0<x1<x2<x3<. . . <xn are successively greater measured amounts of analyte.

y=the chemiluminescence measured for an amount of analyte, representedby x, where y0<y1<y2<y3< . . . <yn are successively greater values ofchemiluminescence, measured for x0<x1<x2<x3< . . . <xn, respectively.

x0=a zero amount of analyte or the amount of analyte equal to zero.

y0=the chemiluminescence measured for an amount of analyte equal tozero, which is x0.

s=one standard deviation of y0.

Then the sensitivity=xn for yn>y0+2s, when n=the least, positive,nonzero integer.

High quantum yield acridinium compounds enhanced the sensitivity of theBayer Diagnostics ACS:180® TSH3 Assay 1.5- to 7.5-fold when compared tothe control acridinium compound NSP-DMAE-HEG-glutarate. The exampleestablishes that when used as chemiluminescent immunoassay labels theenhanced chemiluminescent light emission from high quantum yieldacridinium compounds enhances the sensitivity of sandwich immunoassays.

DEFINITIONS

Analog: A chemical compound with some structural similarity to anotherdifferent compound.

Analyte: A substance or substances in a sample to be identified,assessed, characterized, detected or measured in an assay.

Assay: The method, the reaction or the act of qualitative orquantitative identification, assessment, characterization, detection ormeasurement of the properties or amount of a substance, substances,analyte or analytes or their parts; and is synonymous with test.

Antigen: A substance which elicits the production of antibodies, or animmune system response, generally against a specific determinant ordeterminants.

Antibody (immunoglobulins, immune gamma globulin, immune globulin,immune serum globulin, immunoglobulin): a protein which can normallybind antigen thus producing an immune response.

Assay Reaction: the action of assay reagents on a sample in an assay.

Assay Reaction Mixture: a mixture of sample and assay reagents in anassay or assay reaction.

Carrier: A substance, generally a macromolecule, conjugated or complexedto one or more antigens, atoms, compounds, epitopes, haptens, ions,labels, ligands, molecules, radicals or receptors.

Competitive Immunoassay (generally synonymous with Competitive BindingAssay, Ligand Assay, Ligand-receptor Assay, Hapten Assay, SaturationBinding Assay): A subset of immunoassay where the amount of receptor isless than the amount of ligand and is based on the competitive binding,between a labeled ligand (tracer) and an unlabeled ligand (analyte) fora receptor, or based on the competitive binding between an immobilizedligand and a non-immobilized ligand (analyte) for a labeled receptor.

Complex: a non-covalent union of one or more atoms, molecules,compounds, ions or radicals with one or more atoms, molecules,compounds, ions or radicals.

Complexation: formation of a complex.

Conjugate: the act of or a covalent union of a molecule or molecules,compound or compounds with one or more atoms, molecules, compounds, ionsor radicals forming a new molecule.

Conjugation: formation of a conjugate.

Determinant: a specific molecule or feature on the surface of a microbeor macromolecule that triggers an immune response.

Epitope: a specific site on a macromolecule to which a specific antibodycan bind.

Hapten: a small molecule which cannot itself initiate an immune responsebut can act as an antigen when complexed or conjugated to a largercarrier and can thus initiate specific antibody production to which itcan subsequently be bound.

Heterogeneous Immunoassay: immunoassay method which includes steps forseparation of bound substances form unbound substances.

Homogeneous Immunoassay: immunoassay method which has no steps forseparation of bound substances form unbound substances.

Immunoassay: the method, the reaction or the act of qualitative orquantitative identification, assessment, characterization, detection ormeasurement of the properties or amount of a substance or an analyte orits parts by action of an antigen, epitope, hapten, ligand, carrier, ormacromolecule with a receptor which is usually an antibody or otherbiological receptor; and is synonymous with immunologic assay orimmunochemical assay.

Label: an atom, molecule, compound, ion, radical or macromolecule thatwhen conjugated or complexed to a ligand or to a receptor enablesqualitative or quantitative identification, assessment, detection ormeasurement of an analyte in an assay.

Ligand: an atom, molecule, compound, ion or radical that bindsspecifically to a macromolecule (e.g. a hormone is a ligand for itsreceptor).

Macromolecule: a large molecule generally with a molecular weightgreater than 1,000 daltons.

Sample: biological material which potentially contains an analyte oranalytes.

Tracer: an immunoassay reagent made of a conjugate or complex of a labelwith a ligand or a receptor, which by action of the label, enablesqualitative or quantitative identification, assessment, detection ormeasurement of an analyte in an assay.

Receptor: a macromolecule which can bind specifically one or moreantigens, atoms, compounds, epitopes, haptens, ions, labels, ligands,molecules, radicals, other receptors or tracers.

1. A reagent for the detection or quantification of an analytecomprising a hydrophilic, high quantum yield acridinium sulfonamidecompound attached to an analyte, an analyte analog, or a bindingmolecule for an analyte; wherein the acridinium sulfonamide compoundcomprises a sulfonamide linkage at C-9 position of the acridinium ringand an electron-donating functional groups of the form —OR*, where R* isa group comprising a sulfopropyl moiety or ethylene glycol moieties, ora combination thereof, at the C-2 and/or the C-7 position of theacridinium ring, wherein the high quantum yield acridinium sulfonamidecompound has a relative quantum light yield greater than 1 as comparedwith a corresponding acridinium sulfonamide compound withoutelectron-donating functional groups at the C-2 and/or the C-7 positionof the acridinium ring.
 2. The reagent of claim 1, wherein the electrondonating functional group at the C-2 and/or the C-7 position is—OCH₂CH₂CH₂SO₃ ⁻ or —O(CH₂CH₂O)_(n)—CH₂—CH₂—OMe, wherein n=0-5.
 3. Thereagent of claim 1, wherein said acridinium compound comprises saidelectron-donating functional groups at the C-2 and the C-7 position ofthe acridinium ring.
 4. A heterogeneous immunoassay for thequantification of a macromolecular analyte comprising: a) providing aconjugate of a binding molecule specific for a macromolecular analytewith a hydrophilic, high quantum yield chemiluminescent acridiniumsulfonamide compound comprising a sulfonamide linkage at C-9 position ofthe acridinium ring and containing electron donating functional groupsof the form —OR*, where R* is a group comprising a sulfopropyl moiety orethylene glycol moieties, or a combination thereof, at the C-2 and/orC-7 position of the acridinium ring; b) providing a solid phaseimmobilized with a second binding molecule specific for saidmacromolecular analyte; c) mixing the conjugate, the solid phase and asample suspected of containing the analyte to form a binding complex; d)separating the binding complex captured on the solid phase; e)triggering the chemiluminescence of the binding complex of d) by addingchemiluminescence triggering reagents; f) measuring the amount of lightemission with a luminometer; and g) detecting the presence orcalculating the concentration of the analyte by comparing the amount oflight emitted from the reaction mixture with a standard dose responsecurve which relates the amount of light emitted to a known concentrationof the analyte.
 5. A heterogeneous immunoassay for the quantification ofa small molecule analyte comprising: (a) providing a conjugate of ananalyte or an analyte analog with a hydrophilic, high quantum yieldchemiluminescent acridinium sulfonamide compound comprising asulfonamide linkage at C-9 position of the acridinium ring andcontaining electron donating functional groups of the form —OR*, whereR* is a group comprising a sulfopropyl moiety or ethylene glycolmoieties, or a combination thereof, at the C-2 and/or C-7 position ofthe acridinium ring; (b) providing a solid phase immobilized with abinding molecule specific for the analyte; (c) mixing the conjugate,solid phase and a sample suspected of containing the analyte to form abinding complex; (d) separating the binding complex captured on thesolid phase; (e) triggering the chemiluminescence of the binding complexof d) by adding chemiluminescence triggering reagents; (f) measuring theamount of light with an luminometer; and (g) detecting the presence orcalculating the concentration of the analyte by comparing the amount oflight emitted from the reaction mixture with a standard dose responsecurve which relates the amount of light emitted to a known concentrationof the analyte.
 6. A heterogeneous immunoassay for the quantification ofa small molecule analyte comprising: (a) providing a solid phaseimmobilized with an analyte or an analyte analog; (b) providing aconjugate of a binding molecule specific for the analyte with ahydrophilic, high quantum yield chemiluminescent acridinium sulfonamidecompound comprising a sulfonamide linkage at C-9 position of theacridinium ring and containing electron donating functional groups ofthe form —OR*, where R* is a group comprising a sulfopropyl moiety orethylene glycol moieties, or a combination thereof, at the C-2 and/orC-7 position of the acridinium ring; (c) mixing the solid phase, theconjugate and a sample suspected containing the analyte to form abinding complex; (d) separating the binding complex captured on thesolid phase; (e) triggering the chemiluminescence of the binding complexof d) by adding chemiluminescence triggering reagents; (f) measuring theamount of light with an luminometer; and (g) detecting the presence orcalculating the concentration of the analyte by comparing the amount oflight emitted from the reaction mixture with a standard dose responsecurve which relates the amount of light emitted to a known concentrationof the analyte.
 7. A hydrophilic, high quantum yield acridiniumsulfonamide compound having the following structure:

wherein, R₁ is an alkyl, alkenyl, alkynyl or aralkyl, wherein R₁comprises up to 20 heteroatoms; R₂ and R₃ are the same or different andare hydrogen, halides or R where R is an alkyl, alkenyl, alkynyl, aryl,or aralkyl, wherein R comprises up to 20 heteroatoms at positions otherthan C(2) and C(7); W₁ and W₂ are the same or different and representhydrogen or an electron-donating group of the form —OR*, where R* is agroup comprising a sulfopropyl moiety or ethylene glycol moieties, or acombination thereof; with the proviso that at least one of W₁ and W₂ issaid electron-donating group; A⁻ is a counter ion to pair with thequaternary nitrogen of said acridinium nucleus; Y is a group —R₉—R₁₀,where R₉ is not required or is branched or straight-chained alkyl,substituted or unsubstituted aryl or aralkyl, each comprising up to 20heteroatoms, and R₁₀ is a leaving group or an electrophilic functionalgroup attached with a leaving group selected from the group consistingof:

a halide or —COOH; or R₁₀ is a nucleophilic group —NH—R—NHR wherein R isindependently hydrogen, alkyl, alkenyl, alkynyl, or aralkyl; eachcomprising up to 20 heteroatoms; and Y′ is a substituted orunsubstituted aryl group or branched or straight chain alkyl group. 8.The hydrophilic, high quantum yield acridinium sulfonamide compoundaccording to claim 7 having the following structure:


9. The hydrophilic, high quantum yield acridinium sulfonamide compoundaccording to claim 8 having the following structure:


10. The hydrophilic, high quantum yield acridinium sulfonamide compoundaccording to claim 9 having the following structure:


11. The heterogeneous immunoassay of claim 4, wherein the macromolecularanalyte is selected from the group consisting of proteins, nucleicacids, oligosaccharides, antibodies, antibody fragments, cells, viruses,and synthetic polymers.
 12. The heterogeneous immunoassay of claim 5,wherein the small molecule analyte is selected from the group consistingof steroids, vitamins, hormones, therapeutic drugs, and small peptides.13. The heterogeneous immunoassay of claim 6, wherein the small moleculeanalyte is selected from the group consisting of steroids, vitamins,hormones, therapeutic drugs, and small peptides.
 14. The heterogeneousimmunoassay of claim 4, wherein the macromolecular analyte is thyroidstimulating hormone.
 15. The heterogeneous immunoassay of claim 5,wherein the small molecule analyte is theophylline.